Appendix A -Base Calculations

A. 1. IO NIC STRENGTH

Ionic strength (/) is a measure of ion concentration, de• and based on Eq. (1.2) fined by the equation

(W] = (1.0 X 10-14)/0.040 = 2.5 X 10- 13

so that where c. is the concentration of ion i, z. is the charge of ' ion i, and L is "the sum ~f." pH = -log[W] = -log(2.5 X 10- 13) = 12.6 = 13 Problem: What is the ionic strength of a O.lM solu• tion of (NH4 ) 2SO4 ?

Solution: Each "molecule" of (NH4) 2SO4 yields two A.3. HE DERSON- H ASSE LBALCH NH; ions and one So~- ion. Hence, EQUATIO N

[NHl l = c 1 = 0.2M; z1 = + 1 [So~ - ]= c2 = 0.1M; z2 = - 2 Problem 1: Calculate the pH of a solution made by mix• 1 I = I/2 ~{ (0.2 X 12) + (0.1 X ( - 2)2]} = 0.3 ing 15.0mlof 1.00 X 10- MK2HP04 and25.0mlof2.00 2 X 10- MKH2P04 . Solution: Molar concentrations of the two salts in the A.2. pH final volume (15.0 + 25.0 = 40.0 ml) are: 1 forK2HP04 : 15.0 X (1.00 X 10- )/40.0 = 0.0375M Problem 1: What is the pH of 0.01 OM HCI, assuming that for KH2P04 : 25.0 X (2.00 X 10- 2)/40.0 = 0.01 25M the HCl is 100% ionized? Substituting these values into the Henderson- Hasselbalch Solution: equation and using pK~ = 7.21 yields

[W] = O.OlOM = 1.0 X 10-2M pH = -log[W] = -log(l.O X 10- 2) = 2.0 (0.0375) pH = 7.21 +log (0.0125) = 7.69

Problem 2: What is the pH of 0.020M Ca(OH)2 , as• suming that it is 100 % ionized? Problem 2: Given that (HA = CH3 - Solution: Since each "molecule" of Ca(OH)2 yields CHOH -COOH; A- = CH3 -CHOH - coo- ) has a two OH- ions, pK~ of 3.86, calculate the percent of lactic acid present in

503 504 APPENDIX A its dissociated form (A-) at a pH that is one unit above its (0.400- x) = 0.065 mol ofK2HP04/liter pK~ (i.e., at pH 4.86). Solution: At pH 4.86, the Henderson-Hasselbalch Since you wish to prepare not I liter but only 500 ml, equation takes the form you will need one-half of the above number of moles,

namely, 0.168 mol ofKH2P04 (22.8 g) and 0.033 mol of [A-] K2HP04 (5.75 g). To prepare the buffer, you would weigh 4.86 = 3.86 + log [HA] out these amounts, dissolve them in water, and dilute the solution to a final volume of 500 ml. Hence, Problem 2: How would you prepare 500 ml of a O.lOOM acetate buffer at pH 5.00 from acetic acid and NaOH? The pK~ of acetic acid is 4.76. Solution: Substituting into the Henderson-Hassel• balch equation yields so that [A-] 5.00 = 4.76 + log[HA] [A-] = 10 = !.Q [HA] 1 Hence, For every mole of HA per liter, there exist 10 moles of A- per liter. The percentage of lactic acid present in the [A-] log [HA] = 0.24 A- form (percent dissociation) is [A-] [A-] 10 [HA] = 1.74 %A-= [A-]+ [HA] X 100 = (10 + 1) X 100 = 91% Since you wish to prepare a O.IOOM buffer, the total, In like manner you can calculate that, at a pH that is combined concentration of A- and HA must be 0.1 OOM, one unit below the pK~ value (pH= 2.86), 91% of the lac• that is, tic acid will be present in its undissociated form (HA). You can also show that at a pH that is two units above (or be• [A-] + [HA] = 0.100 low) the pK~ value, the percentage of lactic acid present in the dissociated (or undissociated) form rises to 99%. Each of the last two equations has two unknowns, [A-] and [HA]. You can solve two equations with two un• knowns simultaneously. From the first equation, you have that

Problem 1: How would you prepare 500 ml of a 0.400M [A-] = 1.74[HA] buffer at pH 6.50 from solid KH2PO 4 and ~HPO 4 ? The second pK~ of H3PO4 is 7.21. Substituting for [A-] into the second equation yields Solution: A 0.400M phosphate buffer contains a to• tal of 0.400 mole of phosphate salts per liter. Hence, let• 1.74[HA] + [HA] = 2.74[HA] = 0.100 ting x equal the number of moles of KH2PO 4 per liter, it follows that (0.400 - x) is the number of moles of so that K2HPO4 per liter. Substituting into the Henderson-Has• [HA] = 0.0365M (0.0365 mol/liter) selbalch equation yields [A-] = 1.74[HA] = 0.0635M (0.0635 mol/liter)

6.50 = 7.21 +log (0.400- x) As you wish to prepare only 500 ml, not 1 liter, you X will need one-half of the above number of moles, name• log (0.400- x) = -0.71 ly, 0.0182 mol of HA (acetic acid) and 0.0318 mol of A• X (acetate). Since both buffer components are derived from acetic acid, you must start with 0.0500 mol of acetic acid (0.400- x) = 0.195 X and convert some of it to acetate by adding NaOH. From the neutralization reaction involved, x = 0.335 mol of KH2PO iliter APPENDIX A 505

0.0500- 0.0318 = 0.0182 mol you see that adding 1.0 mol of NaOH converts 1.0 mol of To sum up, you would prepare this buffer by obtain• acetic acid to 1.0 mol of acetate. To produce 0.0318 mol ing 0.0500 mol of acetic acid (2.87 ml of concentrated of acetate, you must add 0.0318 mol of NaOH. After acetic acid, which is 17 .4M), adding 0.0318 mol of solid adding the NaOH, you will be left with the required NaOH (1.27 g), and diluting the mixture to 500 mi. amount of acetic acid: Appendix B Principles of Organic Chemistry

TIONAL CR LITY

Functional groups consist of two or more and pos• Many objects are asymmetric in their structure; they have sess characteristic structures and chemical reactivities. A a "handedness" like that of the left and right hands. If you given functional group generally behaves the same way in visualize your two hands placed on either side of a flat all molecules containing that group. Table B.l shows mirror, one hand will appear to be the mirror image of the some of the functional groups found in biomolecules. other. But the two hands are not identical. You cannot put one hand on top of the other, with both palms down. The two hands cannot be superimposed in space. B.., POL\ In much the same manner, biomolecules can have structural asymmetry, resulting in mirror images that Polar reactions result from the attractive force between cannot be superimposed in space. We use the term chi• positive and negative charges (or partial charges) on mol• rality ("handedness") to refer to the right- and left-hand• ecules. We call the two reactants in a polar reaction nu• edness of a molecule. At the molecular level, chirality cleophile and electrophile. A nucleophile consists of an arises when a compound contains one or more chiral or a group of atoms that has an electron-rich site and centers. A chiral center comprises either a chiral carbon forms a bond by donating a pair ofelectrons. By contrast, atom or some other asymmetric region in the molecule. an electrophile consists of an atom or a group of atoms A chiral carbon atom has four different substituents at• that has an electron-poor site and forms a bond by ac• tached to it (Figure B.l). Because tetravalent carbon is cepting a pair of electrons. We depict the electron-pair tetrahedral, these groups occupy the corners of a tetrahe• movement by means of a curved arrow, using the con• dron. Because of carbon's tetrahedral nature, the two vention that the electron pair moves from the tail to the mirror images of a chiral carbon cannot be superim• head of the arrow: posed. Aside from carbon, several other atoms that form ...... _ compounds having a tetrahedral structure (Si, N, P, S) can A:-- + s+ ---7 A:B exist as chiral centers under proper circumstances. Chiral Nucleophile Electrophile centers also result from molecular asymmetry that is not ----...... _-- s+ s-:?" due to the presence of chiral atoms. The helical structures provide an example. A helix A:- + B: C ---7 A:B + of and nucleic is intrinsically chiral; a left-handed helix constitutes a The second arrow in the second reaction indicates that nonidentical and nonsuperimposable mirror image of a C leaves, taking the two electrons of the B-C bond right-handed helix, much as a left-handed screw differs with it. from a right-handed screw.

507 508 APPENDIX B

Table 8.1. Some Common Functional Groups in Biomolecules

Compound type Structure Functional group

I Alcohol R-C-OH Hydroxyl I

~0 Aldehyde R-C Carbonyl "R ~0 Ketone R-C Carbonyl Mirror

"R· Figure B. l . The two enantiomers (mirror images) of a chiral carbon ~0 atom. Acid R- C Carboxyl "-oH I c is the concentration of the solution (in grams per 100 Amine R-C-N~ Amino ml). I The two mirror images of a compound containing a chiral carbon, called enantiomers, differ in their optical ~0 Amide R-C Amide rotation; they represent optical isomers. If we separate an equimolar mixture of the two enantiomers, we find that """NH 2 one enantiomer rotates the plane of plane-polarized light to the right; it is dextrorotatory and designated ( + ). The ~0 other enantiomer rotates the plane the same number of de• Ester R-C Ester grees but to the left; it is levorotatory and designated (- ). """0 - R' An equimolar mixture of the two enantiomers has a net I I zero optical rotation and is called a racemic mixture. Ether R- C- 0 - C-R' Ether In addition to enantiomers, two other types of opti• I I cal isomers can occur--diastereomers and meso com• Thiol R- S-H Sulfhydryl pounds. Diastereomers constitute optical isomers of a compound that are not mirror images. For example, the Disulfide R- S-S-R' Disulfide threonine has two chiral carbons(*) and a to• tal of four optical isomers: 8.4. OPTICAL ISOMER! M coo- coo- I I + Chiral compounds are optically active; they exhibit opti• H+ H ~ N-C * - 1-1 11 -C*- 3 cal rotation. When plane-polarized light is passed I I through solutions of chiral compounds, they rotate the 11 -C*- OH HO- C* - H plane of polarization. Most optically active compounds I I encountered in biochemistry owe their activity to the pres• CH, CH 3 ence of one or more chiral carbons. We describe optical L-Threonine o-Threonine rotation in terms of a specific rotation, [a], usually mea• coo- coo- sured at25°C, using light of the sodium D line (589.3 nm): I I + H+ H3N-C*- I! 11 -C*- 3 I I HO - C* - H H- C* - OH I I where a is the observed rotation in degrees, dis the opti• CH3 CH3 cal path length through the solution (in decimeters), and L-o//o-Threonine o-a //o-Threonine APPENDIX B 509

Of these, L- and o-threonine are enantiomers, and so are structures, differing from one another by one or more L-alla- and D-alla-threonine. However, L-and L-alla-thre• slight rotations of atoms about single bonds. A single, onine are diastereomers, and so are o- and o-allo-threo• specific conformational form, or conformer, cannot be nine. isolated. A meso compound contains two or more chiral car• Configuration refers to a unique and fixed spatial bon atoms but has no optical activity because the mole• arrangement of atoms in a molecule such that the mole• cule possesses a plane of symmetry. For example, the bio• cule can be isolated in that particular stereochemical chemically important structure formed by linking two form. The change from one configuration to another re• molecules of the amino acid cysteine via a disulfide bond quires breaking covalent bonds and re-forming them in (often referred to as cystine) can exist in the form of three a different sense. The covalent structure of the molecule isomers: is changed as one configuration is replaced by a nother. Different configurational forms constitute stereoiso• coo- coo- coo- mers. I I I + + H+ H 3 N - C* - 11 H 3 N- C *-H H- C* - .l I I I 8.6. L>, t A D R, 5 SYSTCMS CH2 CH2 CH2 I I I s s We assign specific configurations to enantiomers by ref• I ------1------I erence to glyceraldehyde, CHO-CHOH -CH20H. This s s s as a standard b ecause chemists I I I compound was chosen pure form and CH2 CH2 CH2 were able to isolate its two enantiomers in I I I to determine the precise configuration of each enantiomer. + + NH+ - C* - H - C* - 11 The dextrorotatory enantiomer was designated o, and the 11- C* - 3 HJ H.l I I I levorotatory enantiomer L. In a two-dimensional projec• coo- coo- coo- tion, in which we write the structure of glyceraldehyde as L-enantiomer meso compound o-enantiomer shown below, the D-isomer has the H ofth e chiral carbon on the left. In the n, L system, we designate optically ac• The meso compound has a plane of symmetry that tive compounds, such as amino acids and , passes through the disulfide bond, at right angles to the o or L by matching up their structures with that of glycer• axis of this bond. Because of the plane's bisection, one aldehyde. part of the meso compound is identical to half of the levo• rotatory enantiomer, while the other is identical to half of CHO CHO the dextrorotatory enantiomer. Thus, half of the meso compound tends to rotate the plane of plane-polarized light to the right, while the other half tends to rotate it the same number of degrees to the left. The two parts produce an internal compensation, making the molecule, as a CHpH CHp H whole, optically inactive. n-Glyceraldehyde L-Glyceraldehyde

For molecules other than glyceraldehyde, there ex• .5. O N FORMATIO ists no necessary correspondence between configuration AND CONFIGU ATIO (o or L) and optical rotation ( + or - ). Originally, the terms o and L were meant to indicate the direction of ro• Conformation refers to the spatial arrangement of atoms tation, dextrorotatory and levorotatory. Moreover, o-glyc• in a molecule resulting from their freedom of rotation eraldehyde and many other o-monosaccharides are indeed about single bonds. Production of different conforma• dextrorotatory, o( + ).However, many other o-compounds tions involves no change in the covalent structure of the are levorotatory, o(- ). Likewise, there occur both levo• molecule; the change from one conformation to another rotatory L-compounds, L( - ), and dextrorotatory L-com• does not require breaking and remaking of covalent pounds, L( + ).The magnitude and direction of optical ro• bonds. When we speak of the conformation of a mole• tation are complicated functions of the electronic structure cule, we are, in reality, dealing with an entire family of surrounding the chiral center. 510 APPENDIX 8

The need for a reference compound, as in the n, L ing "left"). Let us illustrate the convention for glycer• system, is eliminated when we use the R, S system. By aldehyde: means of this system, we can assign absolute configura• tions to any compound by examining its stereochemical H OH 4 4 HO H \ jJ. structure. c ( ""cl In the R, S system, we assign a priority to each group I \ ) I \ attached to a chiral carbon by using a set of rules. We then 2 HOCH2 CHO 3 3 CHO CHzOH 2 examine the structural formula with the group of lowest ~ "-.../ priority facing away from the viewer. If the priority of the D-Glyceraldehyde L-Glyceraldehyde remaining three groups decreases in a clockwise order, = R-Glyceraldehyde = S-Glyceraldehyde we designate the absolute configuration as R (from the Latin rectus, meaning "right"). If the priority of the Despite the advantages of the R, S system, the D, L groups decreases in a counterclockwise order, we desig• terminology is still commonly used in biochemistry and is nate the configuration asS (from the Latin sinister, mean- employed in this book. Appendix C Tools of Biochemistry

Typical spectrophotometric assays require construc• tion of a standard curve-a plot of absorbance as a func• Many compounds of biological interest absorb light in tion of concentration for several standard solutions of a the ultraviolet, visible, or near-infrared regions of the compound. Drawing the line of best fit through the points spectrum. When radiation is absorbed by a substance, the allows us to determine the concentration of an unknown energy of the radiation raises the substance from one en• from its absorbance and the standard curve. ergy level to a higher one; the substance undergoes a tran• sition. Different wavelengths of radiation have different energies associated with them and cause different types of transitions. We determine absorption of light by pass• ing it through a solution and measuring the incident and Chromatography comprises a group of methods for sepa• transmitted intensities in a spectrophotometer. These rating complex mixtures of molecules based on their repet• measurements yield the absorbance (A), defined by itive distribution between a mobile and a stationary phase. Beer's law: Distribution of molecules between the two phases is gov• erned by one or more of four basic processes: adsorption, A = !og(Ijl) = Elc ion exchange, partitioning, and gel filtration. Movement of the mobile phase results in a differential migration, or where /0 is the intensity of the incident light, I is the in• resolution, of the molecules along the stationary phase. tensity of the transmitted light, c is the concentration of the absorbing substance, l is the length of the light path through the solution, and E is the extinction coefficient. The extinction coefficient represents the absorbance In adsorption chromatography, the stationary phase of a solution when the concentration and the length of the consists of a solid, typically in the form of a column (Fig• light path are both unity. Because absorbance is a dimen• ure C.l ), and the mobile phase is an aqueous or nonaque• sionless quantity, the units of the extinction coefficient de• ous solution. We apply the sample to the top of the col• pend on l and c and must be such that Elc has no units. If umn, develop the column by passing a suitable liquid c represents a molar concentration, we call E a molar ex• through it, and collect the effluent, frequently by means of tinction coefficient (E). We can also express absorption of an automatic fraction collector. Components are eluted light in terms of the fraction or the percent of incident light when they emerge in the effluent. As the material moves transmitted, called transmittance (T) or percent trans• down the column, adsorption and desorption occur re• mittance (%T), respectively: peatedly. Adsorption involves van der Waals forces, hy• drogen bonds, and hydrophobic interactions. The rate of

T = 1/10 %T = (///0) X 100 movement of a substance through the column depends on

511 512 APPENDIX C

Column Sample Column Elution of preparation application development components

t i

Fractions collected

r 1gure C. 1. Column chromatography. Concentration in the effluent is measured as a function of fraction number or elution volume. the degree of adsorption of the substance to the stationary cross-linked polymer having many charged functional phase. A substance that adheres more strongly to the col• groups. A cation-exchange resin has a negative charge and umn will move more slowly through it. binds cations; an anion-exchange resin has a positive charge and binds anions. The mobile phase consists of an C.2.2. !on-Exchange Chromatography aqueous solution. Ions in the sample bind electrostatical• ly to oppositely charged groups of the resin (Figure C.2). In ion-exchange chromatography, the stationary phase Subsequently, we disrupt these ionic bonds by changing (typically, a column) consists of an ion-exchange resin, a the pH and/or ionic strength of the eluting buffer. Binding

A B c

Figure C. 2 · Ion-exchange chromatography. (A) A cation-exchange resin with bound Na+ ions. (B) Amino acid cations replace Na+ on the col• umn. (C) Changing the pH and/or the ionic strength of the eluting buffer alters the charges of the bound amino acid ions and leads to their elution. APPENDIX C 513 and desorption of ions occur repeatedly as the material same general shape. Likewise, more compact and globular moves down the column. The extent to which ions are re• molecules diffuse more rapidly than elongated, asymmetric tarded in their movement through the column depends on ones of the same size. The more extensive is the penetration the sign and magnitude of their charge. of molecules into the beads, the more retarded their move• ment through the column and the greater the elution volume. We can use gel filtration for molecular weight determina• tions since, for proteins of the same general shape and for a In molecules distribute partition chromatography, given gel, a linear relationship exists between the elution themselves repeatedly between two immiscible phases ac• volume and the logarithm of the molecular weight. cording to their in these phases. In paper A special type of chromatography, called affinity chromatography (Figure C.3), the stationary phase com• chromatography, relies on the specific noncovalent bind• prises a polar layer of water molecules bound to the hy• ing (by adsorption and/or ion exchange) of a ligand to the droxyl groups of cellulose in the paper. The mobile phase molecule of interest (Figure C.5). The ligand is linked co• usually consists of a relatively nonpolar organic solvent valently to a column, and the technique leads to extensive that moves over the stationary aqueous phase. In such a purification of the molecule of interest in a single step. system, nonpolar molecules move faster along the paper Frequently, researchers perform chromatographic than polar ones. In descending chromatography, material separations using special columns through which the mo• moves down the filter paper by gravity; in ascending chro• bile phase is forced under high pressure (5000-10,000 matography, it moves up by capillary action. psi). Such high-performance liquid chromatography We characterize the movement of substances by Rf (HPLC) requires small amounts of sample, is rapid, and values, which are generally proportional to solubilities in achieves high resolution. Typically, peaks are much sharp• the mobile phase. An Rf value is the ratio of the distance er than those shown in Figure 2.6. moved by the sample to that moved by the solvent, both measured from the point of sample application, or origin:

R = _di_s_ta_n_ce_m_o_v_ed_by"--sa_m_p!...l_e f distance moved by solvent We can separate macromolecules in solution by subjecting them to a centrifugal force, generated by spinning a rotor about its axis. The centrifugal force causes macromole• cules to sediment through the solution and leads to their In gel-filtration chromatography, the stationary phase separation based on differences in molecular size, shape, consists of spherical gel particles (beads) of controlled size and density. We use preparative centrifuges with fixed-an• and porosity, formed from cross-linked polymers (Figure gle rotors primarily for purification and fractionation of C.4). Molecules are fractionated on the basis of their size macromolecules. Analytical ultracentrifuges are equipped and shape, two properties that determine the rate of diffu• with optical systems that allow photographs and measure• sion. Smaller molecules diffuse faster than larger ones of the ments to be made during sedimentation (Figure C.6).

-~

Solvent front (R1 = 1)

Sample spot -- "E Q) E a • Q) > 0 E b "E Q) > 0 Figure C.l. Principle of paper chromatogra• (f) phy. Typically, ascending chromatography em• Origin (R1= 0) ploys a filter paper cylinder placed in a glass jar -- having solvent at the bottom. In descending chro• matography, a filter paper sheet is hung from a solvent trough in a rectangular glass chamber. Filter paper / Proteins ! ••••• •• • • • Gel particle oo••••• (bead) oo oo l ____f ••• A 8 ••c Figur C.4. Gel filtration. Smaller molecules penetrate the gel beads and are retarded; larger molecules are eluted first.

Crude mixture D o

D o 0 D 0 0 0 0 Purified component A 8 c

Figur C.S. Principle of affinity chromatography. A ligand, linked covalently to a column, binds a specific . Changing the eluting buffer disrupts the ligand-protein interactions, yielding a highly purified protein preparation. APPENDIX C 515

A Axis of rotation

Centrifuge tube

Pelleted material

B Mirror _/ Viewing /,-- Optical system--- Photographic film · Recorder Motor

+--- Steel chamber Rotor Sample compartment

Light source V

Figure C.6. Centrifugation. (A) A fixed-angle rotor; (B) diagram of an analytical ultracentrifuge.

C.3 .1. Analytical Ultracentrifugation particle (mllg), and p is the density of the solution (g/ml). In the analytical ultracentrifuge, each sedimenting com• ponent forms a boundary, a transition between solvent and C.3.2. Density Gradient Centrifugation solution or between two solutions. An optical system de• tects the boundary and causes it to appear as a peak on a In density gradient centrifugation, sedimenting parti• photograph (Figure C.7). We measure the movement of cles move through a density gradient, a liquid column in this peak as a function of time by determining its position which the density increases from top to bottom. When we on successive photographs. From these measurements, we perform density gradient centrifugation in the analytical can determine the sedimentation coefficient (lower case ultracentrifuge, we dissolve the sample in a dense salt so• s). A sedimentation coefficient- the velocity per unit of lution. The salt distributes itself during centrifugation and centrifugal field- is expressed in terms of Svedbergs thereby sets up a density gradient. Macromolecules of a (capitalS). One Svedberg equals I o- 13 s. We can also use given type band in this gradient at a position where their analytical ultracentrifugation to determine molecular density equals that of the gradient. We determine the den• weights (M) of sedimenting particles by means of the sity of material in a band from the band's position. When Svedberg equation: we perform density gradient centrifugation in a prepara• tive ultracentrifuge, we place the sample on top of a pre• M = (RTs)ID(l - Vp) formed density gradient, prepared by successively layer• ing solutions of different densities in a centrifuge tube. We where R is the gas constant, T is the absolute tempera• then place the tubes in a swinging-bucket rotor, where they ture, s is the sedimentation coefficient, D is the diffu• swing out during centrifugation and line up at right angles sion coefficient, V is the partial specific volume of the to the axis of rotation (Figure C.8). 516 APPENDIX C

Sedimentation

de dx

X

Solvent

Time t

Solution 1

• • • • •• • • • • • • • • • • •• • Time • • zero •• • • •••

• • Solution of • • • macromolecules Figure C.7. Formation of ultracentrifuge patterns • by light passing through a solution of sedimenting particles. Photographs provide plots of concentration gradient (dc/dx) as a function of distance (x) from the Light center of rotation, and boundaries appears as peaks.

A- Slow oompo"'"' g:._ Fast component

Axis of rotation Axis of rotation Axis of rotation A B c r igur<' C. H. Density gradient centrifugation with a swinging-bucket rotor. (A) Loading of sample on top of the gradient. (B) Components sepa• rate during centrifugation. (C) The gradient prevents mixing by convection at the end of the run. Tubes can be punctured at the bottom, and frac• tions collected and analyzed. APPENDIX C 517

C.4. ELECl ROPHORESIS column of a gel (gel electrophoresis). Upon application of an electric field, sample components migrate as spots or Electrophoresis represents the movement of a charged zones (Figure C.9). Polyacrylamide gel electrophoresis particle in an electric field. The velocity, v, of the particle (PAGE) is currently the most popular type of zone elec• is given by trophoresis and utilizes gels made by polymerizing and cross-linking acrylamide (CH2 =CH-CO-NH2 ) . In• v = Eq!f vestigators control pore size in the resultant network by varying the concentration of acrylamide. Biochemists use where E is the electric field (V /em), q is the net charge of polyacrylamide gel electrophoresis for sequencing nucle• the particle, and f is the frictional coefficient. ic acids and for analysis of proteins, as described below. The frictional coefficient depends on particle size In disc gel electrophoresis, discontinuities of pH, and shape. If E is constant, the velocity becomes a func• ionic strength, buffer composition, and gel concentration tion of q!f, the charge-to-mass ratio of the particle. Of two are built into the gel column. Such gels permit the separa• particles having the same mass and shape, the one with tion of a large number of closely related proteins with high greater net charge (q) will move faster in electrophoresis. resolution and require only small amounts of sample. We Of two particles having the same mass and net charge, the layer the sample on top of the gel column and immerse the one that is more more globular rather than elongated, and two ends of the column in buffer compartments contain• hence has a smaller f value, will move faster. We define ing electrodes. After electrophoresis, we stain the gel, and electrophoretic mobility (u) as the velocity per unit of proteins appear as discrete thin bands. electric field: Biochemists use sodium dodecyl sulfate-poly• acrylamide gel electrophoresis (SDS-PAGE) for deter• u = viE= q!f mining molecular weights of individual polypeptide chains. We first dissociate oligomeric proteins into We refer to the major type of electrophoresis em• monomers by treatment with sodium dodecyl sulfate ployed in biochemical research as zone electrophoresis. (SDS), a detergent that breaks hydrophobic bonds, and Researchers perform it by placing a small aliquot of solu• mercaptoethanol, a compound that breaks disulfide tion in contact with some support medium such as a sheet bonds. Under these conditions, proteins yield polypep• of filter paper (paper electrophoresis), a strip of cellulose tide chains that form random coils and that bind large acetate (cellulose acetate electrophoresis), or a slab or amounts of SDS. The charges of the SDS- protein com-

Filter paper Sample or gel slab Initial I ! Anode t •• Cathode 0 •••••• •• 0 •••••• •• ••Origin ••

Final •• • ••: ••• • 0 .: •••• v •• Migrated spots or zones

r igure C ' . Zone electrophoresis. In the illustration, both components carry a net positive charge at the pH of the experiment. 518 APPENDIXC

Table C.1. Some Isotopes of Biochemical Importance

Isotope Radiation emitted Half-life

3H J3 12.3 years l4C J3 5568 years 24Na J3 , 'Y 15 hours 32p J3 14.2 days Jss J3 87.1 days

The half-life (t112) is the time required for the activ• ity of a population of radioactive atoms to decrease by one-half (N = Nof2) and can be computed from

t112 = In 2/'A = 0.693/A. Distance migrated ---. Table C.llists half-lives of some isotopes commonly used r igu re . I 0 . Determination of molecular weights (M) by SDS-PAGE. in biochemical research. Isotopes differ in the energy of the emitted particle. Thus, tritium eH) and 32P represent plexes are effectively due to the bound SDS alone since weak and strong 13 emitters, respectively. we minimize protein charges by performing the reaction The basic unit of radioactivity, the curie (Ci), equals at pH 7. Because all proteins bind essentially the same 2.2 X 10 12 disintegrations per minute (dpm). Because amount of SDS per gram of protein, all SDS-protein we do not normally detect every disintegration by the in• complexes have essentially the same charge/mass ratios strument used, we call events actually measured counts and therefore the same electrophoretic mobility. When per minute (cpm). We measure radioactivity by means of we subject these complexes to electrophoresis on a poly• three basic methods: autoradiography, Geiger-Muller acrylamide gel, they are separated by the sieving action counting, and liquid scintillation counting. of the gel. The gel retards large molecules more than In autoradiography, we expose films with special smaller ones so that large molecules move more slowly emulsions, by means of various techniques, to the radia• through the network. Migration distances are inversely tion emitted by radioactively labeled compounds. Ex• proportional to the logarithm of the molecular weight posed silver halide grains in the film indicate locations of (Figure C.l 0). By calibrating the gel, using migration dis• the radioactive isotope in the sample. In Geiger-Muller tances of standards, we can determine molecular weights counting, 13 particles emitted by radioactive isotopes of unknown proteins. cause ionization in a gas by ejecting electrons from gas atoms, thereby producing ion pairs (ejected electron plus residual cation). The electrons and cations produced are C.S. RADIOACTIVITY collected by electrodes and are recorded as a pulse of charge or current by means of a Geiger counter. In liquid Radioactive isotopes are atoms that have unstable nuclei scintillation counting, 13 particles emitted by radioactive and that disintegrate spontaneously to produce nuclei of isotopes excite solvent molecules that emit photons as different elements. Radioactive disintegration, or decay, they return to the ground state. Added compounds, called is accompanied by emission of one or more types of ion• jluors, absorb these photons and re-emit the absorbed en• izing radiation such as a particles (helium nuclei), f3 par• ergy in the form of photons of longer wavelength by ticles (electrons originating in the nucleus), or r rays fluorescence. Fluorescence of the fluors produces flashes (high-energy photons originating in the nucleus). Ra• oflight, or scintillations, which are converted to electrical dioactive decay constitutes an exponential process: pulses and counted.

C.6. RECOMBINANT DNA where N is the number of radioactive atoms present at time TECHNOLOGY t, N is the number of radioactive atoms present at time zer~ (t = 0), A. is a decay constant characteristic of the iso• Recombinant DNA technology , or genetic engineering, tope, and t is time. comprises various techniques by which researchers carry APPENDIX C 519 out genetic recombination in vitro. This technology, initi• tigators then link the annealed covalently by means ated in the early 1970s, includes breaking and rejoining of of DNA (Figure C. II). DNA molecules from different and production Investigators can also generate cohesive ends syn• and isolation of modified DNA or fragments thereof. An thetically by using the terminal deoxynucleotidyl example of recombinant DNA technology is the insertion . This enzyme catalyzes addition of a tail of nu• of a human gene coding for insulin into the DNA of a cleotides to the 3' -end of DNA and does not require a tem• bacterial followed by cloning of the plasmid to plate. Thus, we can add a tail of poly( C) to one DNA and produce many identical copies of the inserted gene. The a tail of poly( G) to another, anneal the two DNAs via their modified plasmid DNA is called a recombinant DNA. synthetic cohesive ends, and link them covalently by Recombinant DNA technology generally involves the fol• means of DNA ligase (Figure C.l2). lowing major steps. C.6.2. Production of Recombinant DNA C.6.1. Se lection of Target DNA Fragment The target DNA fragment, or passenger, is linked to some Researchers usually produce a specific DNA fragment other DNA molecule, termed a vector. Linking may be ac• containing the gene(s) of interest by means of restriction complished by annealing the cohesive ends of the two , preferably those that lead to formation of stag• molecules, followed by action of DNA ligase. Alterna• gered cuts. The cohesive ends thus formed are useful for tively, researchers employ other methods to splice the pas• annealing this DNA fragment to some other DNA mole• senger to its vector. Any molecule that has a replication cule, produced with the same restriction enzymes. lnves- origin and that can replicate after it enters a suitable cell

3' 5' GAATTC 3'

5' 3' CTTAAG 5' t Restriction enzyme DNA 1 EcoRI DNA2 l 5' IAAI'Ii:i I 3' 5' G AAT TC 3'

3' "-----=-..:.....:..: 5' 3' C T T~ ~ 5'

Cohesi~ve ends Cohesive ends Separation of restriction fragments l 5' .!::A'-"A,_,Tc...T,_,C"--__3' I Annealing l 5' AATTC 3'

3' G 5' I DNA ligase l 5' t gjAAT TC 3'

3' G 5' Recombinant DNA

Fi gur C.ll . Production of recombinant DNA by means of restriction enzymes. 520 APPENDIXC

DNA 1 DNA2 5' 3' 5' 3'

3' 5' 3' 5'

Terminal deoxynucleotidyl transferase !~~

5' !'"" - 3' 5' 3'

3' 3' 5' 1 '1 Poly (C) Poly (C) Poly (G) Poly (G) tail tail tail tail

1. Anneal 2. DNA ligase

5' 3'

3' 5'

Poly (G)

Recombinant DNA

Figure C.l2. Production of recombinant DNA by means of terminal deoxynucleotidyl transferase.

(e.g., or phage DNA) can be used as a vector cloning. In biology, a clone defines a group of genetical• (Figure C.l3). ly identical organisms, derived from a common ancestor. Plasmids, extrachromosomal genetic elements of In molecular biology, a clone defines a population of , are circular double-stranded DNA molecules that identical molecules, derived by replication of a common replicate independently of the bacterial chromosome. ancestor. Plasmids generally confer some evolutionary advantage onto their host cell. Thus, a plasmid may carry a marker C.6.4. Selection of Cells Containing for antibiotic resistance or for production of colicins, pro• Cloned DNA teins that are bactericidal to other strains. A bacterium containing such a plasmid shows resistance to certain an• Since not all cells in a culture contain cloned DNA, cells tibiotics or a capacity to produce colicins and protect it• containing such DNA must be isolated by screening the self against other bacteria. entire culture. In the case of bacteria, screening can be done by using a plasmid that confers suitable properties C.6.3 . Insertion of Recombinant DNA on the host cell. The plasmid may, for example, confer re• into Host Cells sistance to specific antibiotics or eliminate the need for specific nutritional requirements. Cells containing the Plasmids and bacteriophages constitute suitable vectors cloned gene can then be detected by growing the entire for transferring target DNA to host bacterial cells. Bio• culture in the presence of the particular antibiotic or in the chemists use other systems for insertion of recombinant absence of the particular nutrient. Another method of DNAs into eukaryotic cells. Because recombinant DNA screening consists of treating the bacterial culture with an• self-replicates independently of the host cell chromo• tibodies against the of the cloned gene. some, many replications of the vector and its passenger can occur for any single replication of chromosomal C.6.5. Polymerase Chain Reaction DNA This provides for extensive amplification of the target DNA. Production oflarge numbers of copies oftar• The polymerase chain reaction (PCR) was introduced in get DNA in any single cell constitutes molecular 1987 and permits amplification of sequences APPENDIX C 521

original DNA serving as template. Action of DNA poly• / SC:..- Gene(s) of interest merase produces new copies of the target segment. Cohesive ends ~ We follow this set of reactions by a second cycle of heat denaturation, annealing, and primer extension. Us• Cohesive ends ~ ing a thermostable DNA polymerase ensures that the en• zyme is not denatured during the heating step and need Plasmid ..--- not be replenished for each cycle. We can obtain such - Antibiotic resistance thermostable enzymes from thermophiles, bacteria that 0 gene (marker) grow at high temperatures and that occur in hot springs and geysers. We repeat the cycle of denaturation, anneal• ing, and primer extension many times. At each cycle, the CD! amount of the DNA of interest is roughly doubled, there• by providing exponential amplification of the target 0 DNA. Ho~oell DNA ~ j ~Foreign DNA 3' 5'

- Target sequence ~~Marker Plasmtd ® 5' 3' ~ Step 1 - Heat denaturation Step 2 - Anneal with flanking primers 3' 1 s· 01 5' t

~ 3 Primers ...... __ ·~ ...... 3' I ®l 5' 5' 3' ~ ~ Step 3 - DNA polymerase l Figure C.IJ. Cloning of DNA in bacteria by means of plasmid vec• I I tors. (I) Insertion of passenger into vector- formation of recombinant DNA from target DNA fragment and plasmid; (2) insertion of recombi• nant DNA (altered plasmid) into host cells; (3) selection of host cells containing recombinant DNA; (4) intracellular replication of plasmids ~r· I \ (cloning); (5) repeated cellular divisions (cloning). I \..

I 1 I' without cloning (Figure C.l4). The method requires a knowledge of the sequences that flank the tar• Step3' get segment in the DNA of interest. Oligonucleotides complementary to these flanking regions are produced synthetically and used as primers for a series of repetitive steps involving DNA polymerase. To carry out the polymerase chain reaction, we heat• I1 I! I denature a sample of DNA containing the target segment ! to separate the two strands. Next, we add the synthetic etc. primers and anneal the DNA strands to the primers. We then use DNA polymerase to extend the primers, with the Figure C.14. Principle of the polymerase chain reaction. Appendix D Oxidation-Reduction Reactions

D . I . I 1;\ Ll- 'S D.2. l)lf

Oxidation-reduction (or redox) reactions involve changes In determining the direction of an overall reaction, keep in the electronic structure of atoms and molecules. Oxi• in mind the sign convention for reduction potentials. Ac• dation represents a loss of electrons (e-), and reduction a cording to this convention, a half-reaction having a high• gain of electrons. Electron losses and gains result from di• ly negative reduction potential involves a strong reducing rect transfer of electrons from one substance to another or agent; one having a highly positive reduction potential in• from transfer of electrons in association with an H, an 0, volves a strong oxidizing agent: or some other atom. Because hydrogen generally acts as an electropositive element and oxygen as an electronega• Reduction potential (volts) tive element, gain of hydrogen and loss of oxygen are equivalent to reduction, and loss of hydrogen and gain of (-) 0 (+) oxygen are equivalent to oxidation. Most biochemical ox• Strong reducing agent Weak reducing agent (weak oxidizing agent) (strong oxidizing agent) idation-reduction reactions involve gain or loss of hydride ions (H- or H:) and are catalyzed by . Relatively few reactions-though some very important Of two half-reactions, the one having the smaller ones-involve gain or loss of oxygen and are catalyzed by reduction potential involves a stronger reducing agent oxidases. than the other half-reaction. Consequently, the half• Table D.l gives some examples of oxidation-reduc• reaction having the smaller reduction potential pro• tion reactions. Each reaction is written as a reduction and ceeds as an oxidation (loss of electrons) and must be represents a half-reaction, one-half of a complete chemi• written in the reverse sense (Figure D.2). The electrons cal reaction. An overall reaction consists of two half-reac• generated by this half-reaction cause the second half-re• tions, one proceeding as a reduction, and the other as an action to proceed as a reduction (gain of electrons). oxidation. There can never be oxidation without reduc• These relationships hold regardless of the signs of the tion, and vice versa. This holds not only for the overall re• potentials. Thus, of two half-reactions having reduction action but for each half-reaction as well. Even the simplest potentials of -0.52 V and -0.27 V, respectively, the for• half-reaction involves an oxidized and a reduced species mer will proceed as an oxidation, the latter as a reduc• (for example, Ag+ and Ag). The two form a conjugate re• tion. Likewise, of two half-reactions having reduction dox pair, much as A- and HA form a conjugate acid-base potentials of +0.18 V and +0.46 V, respectively, the for• pair. mer will proceed as an oxidation, the latter as a reduc• Two half-reactions must be combined in a way that tion. With this in mind, let us proceed to couple two half• preserves electrical neutrality. There can be no net gain or reactions, once under standard and once under actual loss of electrons (Figure D.l ). conditions.

523 524 APPENDIX D

Table 0 .1. Examples of Redox Reactions

Type of reaction Examples

Gain/loss of electrons Ag+ + e- ;::t: Ag Cytochrome b (Fe3+ ) + e- -=t cytochrome b (Fe2+)

Loss/gain of oxygen Mn04- + 2H20 + 3e- ;:t Mn02 + 40H• NO) + 2H + + ze- <=! N02 + H20

Gain/loss of hydrogen 2C02 + 2H + + ze- <=t H2C20 4 NAD+ + 2H + + 2e- <=t NADH + H+

Oxidation

Cytochrome b (Fe2+) + Ag+ Cytochrome b (Fe3+) + Ag

Reduction i Reductant Oxidant Reducing agent Oxidizing agent Reduced form Oxidized form Electron donor Electron acceptor Substance being oxidized Substance being reduced

Figure 0 .1. Example of an overall redox reaction resulting from the combination of two half-reactions.

D.2.1. Standard Conditions +:t acetaldehyde+ 2H+ + 2e- Eo' = +0.20V Suppose that we wanted to couple the following two half• Eo'= + 0.77V reactions under biochemical standard conditions ( concen• trations are l .OM each). We start out by writing both re• Inspection shows that the overall reaction resulting actions as reductions, since the corresponding E 0 ' values from a combination of these two equations would not be are those of reduction potentials: balanced with respect to electrons. Because the overall re• action has to be electrically neutral, we must multiply the Acetaldehyde + 2H+ + 2e- +:t ethanol Eo' = -0.20V reaction by 2. Multiplication does not change the po• 0 3 Eo' = +0.77V tential because E ' measures the tendency of Fe + to un• dergo reduction to Fe2+. This tendency is the same

0 whether one, two, or a thousand atoms undergo reaction. Comparing the E ' values, we see that the acetalde• 0 We now add the two equations and add the E ' val• hyde half-reaction has a reduction potential that is less than ues likewise: that of the iron half-reaction. We conclude that ethanol con• stitutes a stronger reducing agent than Fe2 + . Hence, Fe3+ Ethanol +:t acetaldehyde+ 2H+ + 2e- Eo'= +0.20V will be reduced to Fe2+ by ethanol, which undergoes oxida• tion to acetaldehyde. Therefore, we write the acetaldehyde 2Fe3+ + 2e- +:t 2Fe2 + Eo' = +0.77V reaction in the reverse sense, as an oxidation. Since we re• versed the reaction we must also reverse the sign of the cor• The overall reaction is

0 0 responding E ' , just as I1G ' for a forward reaction becomes

-I1G0 ' for the reverse reaction. Thus, Ethanol + 2Fe3+ +:t acetaldehyde + 2H+ + 2Fe2+ APPENDIX D 525

Polentiomeler

·0.19 '\'

Elec Iron 0 <±> eclron flow l 1floEl w AQar bridge

~ t--

Negalive Posilive electrode electrode (anode) (cathode)

\!:_'JCiale -+pyruvale 2H+-+ H2

Sample: Relerence: 1M pyruvale/1 M laclale 1M H+/1 aim H2

Potentiomeler rr +0.77 Elec ectron Iron 0 <±> flow1 lfloEl w AQar bridge

~ t-- Posilive Negalive electrode electrode (cathode) (anode)

L- 3 Fe + -+ Fe2+ H2 - 2H+ Sample: Reference: 1M Fe3+/1M Fe2+ 1M H +11 aim H 2

Foflure 0.2. Electrochemical cells for determining biochemical standard reduction potentials. Sample half-reactions are measured at 25°C and pH 7.0 against the standard hydrogen electrode.

0 for which 6.£ ' = ( + 0.20) + ( + 0.77) = + 0.97 V. Be• dized and reduced forms have the following arbitrary 0 0 cause I!J.E ' for the overall reaction is positive, D.G ' for values: the same reaction is negative (Eq. 12.3): [acetaldehyde] = 5.0M [ethanol]= 1.0 X I0- 16M !::.Go' = - (2)(96,491)( + 0.97) = - 187,193 1 mol- 1 = - 187 kJ mol- 1 In order to decide which species, under these condi• We conclude that we have coupled the two half-reactions tions, is the stronger reducing agent, we must calculate the properly under biochemical standard conditions. The biochemical actual reduction potentials by means of the negative 6.G0 ' indicates that the overall reaction proceeds Nemst equation: spontaneously.

E ' _ E o' 0.06 I [reductant] 0.2.2. Actual Conditions - - - 2- og [oxidant] To determine the direction of a redox reaction under non• standard conditions, we have to use biochemical actual, Using this equation, we have for the iron half-reaction rather than standard, reduction potentials. As an exam• ple, assume we wish to couple the two reactions that we ' - 0.06 5.0 E - + 0.77 - log -l.--x- - -_-:-::- = + 0.27 V considered above when the initial concentrations of oxi- 2 0 10 16 526 APPENDIX D and for the acetaldehyde half-reaction You now proceed as before. Reverse the iron half-re• action and change the sign of its potential, the actual re• duction potential. Multiply the iron half-reaction by 2 in E' = -0.20- 0.~6 log 1.00 5~0 w-16 = +0.30 V order to balance the electrons and add the two reactions; add the potentials likewise: Note these important guidelines: E' = -0.27 V You must calculate the actual potentials in order Acetaldehyde + 2H+ + 2e- ~ ethanol E' = +0.30V to decide which species is the stronger reducing agent under nonstandard conditions. You cannot The overall reaction is arrive at this decision by inspecting the values of

the standard reduction potentials. Acetaldehyde + 2Fe2+ + 2H+ ~ ethanol + 2Fe3+ You must use tabulated values of E 01 to calcu• late actual potentials, regardless of the fact that, for which t).E' = ( -0.27) + ( +0.30) = + 0.03 V, and, ultimately, you will reverse one of these half-re• based on Eq. (12.4), actions and change the sign of its potential. You will have to change the sign of the actual poten• 11G' = -(2)(96,491)(+0.03) = -5789 J mol- 1 0 tial; do not change the sign of E '. = -5.8kJmol- 1 You must balance the two half-reactions with re• spect to electrons. In the present example, you You have coupled the two reactions properly, this use n = 2 for both reactions even though the iron time under nonstandard conditions. Because t).E' is posi• half-reaction involves only one electron. Proper tive, t).G' is negative, and the reaction proceeds sponta• coupling requires that the iron half-reaction be neously as written. Note that the overall reaction has been multiplied by 2. reversed from that occurring under standard conditions simply as a result of changes in the concentrations of re• As it turns out, under the conditions chosen, the re• actants and products. As pointed out in Section 9.1, what duction potential of the iron half-reaction is less than that ultimately determines the direction in which a reaction of the acetaldehyde half-reaction. Hence, under these con• proceeds intracellularly are the concentrations of reac• ditions, Fe2+ is a stronger reducing agent than ethanol. tants and products. Consequently, acetaldehyde will be reduced to ethanol by Fe2 +, which undergoes oxidation to Fe3+. Answers to Problems

Because both compounds can form hydrogen bonds with water. Obtain 28.7 ml of concentrated acetic acid, add NaCI dissociates into Na+ and Cl- ions, and soap 178.0 ml of 1.00M NaOH, and dilute the solution to micelles carry a negative surface charge. Because 2.00 liter with water. these particles are charged, water dipoles cluster 63.3. around them. The water shells prevent the solutes pH 6.8. from interacting with each other. The phosphate buffer, because a buffer is most ef• Yes, inverted micelles can form. Their inner core fective within ::'::1.0 pH unit from its pK~. consists of polar carboxyl groups and is surrounded Weigh out 2.11 g of KH P0 and 1.66 g of K HP0 , by a nonpolar shell composed of hydrocarbon 2 4 2 4 and dissolve the salts in 250 ml of water. chains. (a) 0.157; (b) 9.55 mi. (a) 1/2; (b) 1/4; (c) 1/16; (d) 1/64. pH 4.76. -21.8kj. w-4. NaCI can disrupt electrostatic bonds, can Because of the values calculated from the ion prod• disrupt hydrogen bonds, and SDS can disrupt uct of water (Kw = 1.0 X 1 o- 14). When [H+] = 1.0 hydrophobic interactions. Disrupting interactions X 10-14, pH = 14; when [OH-] = 1.0 X 10-14, results from the added compound competing [H+] = 1 .0, and pH = 0. The pH scale can extend with and substituting for the original solute. Urea below 0 and above 14. For example, when [H+] = breaks hydrogen bonds in a protein by forming 2.00M, pH = -0.30; when [OH-1 = 2.00M, [H+] hydrogen bonds with donor and acceptor groups = (1.0 X 10-14)/2.00, and pH = 14.3. in the protein; SDS disrupts hydrophobic interac• The first proton dissociating from each phosphate tions in a biomembrane by binding to membrane group has a pK~ approximately equal to pK~ of ; and ions bind to polar groups of an affected H PO because in both instances dissociation solute. 3 4 be• curs from an uncharged entity. In H P0 , the proton Charge separation occurs along each CO axis 3 4 o+ &- dissociates from an uncharged molecule; in ATP, it (C=O), but the molecule as a whole does not con• dissociates from an uncharged group. stitute a dipole; the centers of the positive and nega• 1.20. pK~ = 6.60. tive charges coincide. 1.21. 0.200M. 1.7. Polar: hydroxymethyl, sulfonic acid, amide. Nonpo• 1.22. , formic acid, and (2). lar: sulfide, phenyl, methyl. Capable of hydrogen 1.23. According to the Henderson-Hasselbalch equation, bonding: hydroxymethyl, sulfonic acid, amide. a buffer's pH is determined by its pK~ value and the 1.8. Water: 1.2 X 10-12 g; protein: 1.3 X 10-13 g; DNA: ratio [A -l/[HA]. For phosphate buffers, pK~ is fixed, 5.3 X 10-15 g; RNA: 1.0 X 10-12 g; polysaccharide: but we can vary the concentrations of A- and HA 2.0 x w-14 g. to produce different buffers. As long as the ratio

527 528 ANSWERS TO PROBLEMS

[A -]/[HAl remains constant, the buffer's pH will re• The order of elution is (first), , aspartic main unchanged even though its molarity will vary. acid (last). 10-5-10-BM. 0 76%. II Any amide bond contains the grouping -C-NH-, but a peptide bond exists only when the CO and NH groups derive from a-carboxyl and a-amino groups of two amino acids. (a) Both amino acids still classified as polar with un• (a) MW = 24,560; (b) four disulfide bonds; (c) 6.35 charged R groups; (b) both amino acids classified as mg. having negatively charged R groups. 27 peptides:

0 AAA + II ppp NH3 -CH2 -CH2 -C-NH-CH-Coo- GGG l CH 2 I AAP,AAG GGA, GGP PPA, PPG C-NH APA, GAA GAG,GPG PAP, PGP PAA, AGA AGG, PGG APP, GPP II )H AGP CH-NH+ APG GAP The equilibrium constants are: GPA PAG PGA (a)

At the isoelectric point the amino acid has an HOOC-CH2 -CH-COOH equal number of positive and negative charges. I Mathematically, we can express this condition by NH! equating the concentrations of the cationic (H 2A+) and anionic (A-) forms of the amino acid. From the I pK' = 2.09 first equation, ~ a1 HOOC -CH2 -CH -coo- [H N] = [HN=][H+]/K' 2 a1 l N~ and from the second equation

I pK' = 3.86 ~ a2

Equating the two terms, -ooc-CH2 -CH-coo- l NH3 + predominates at pH 6 [H+] = (K' K I )1/2 a 1 a2 I pK' = 9.82 pH= -log[H+] = ~ (pK' + pK' ) 3 a1 a 2 ~ a -ooC-CH -CH-Coo- 2.4. pi = 6.68. 2 2.5. There is no pH at which the absolute charge of an l NH ordinary protein is zero; at best, the molecule can 2 have a net zero charge. predominates at pH 12 ANSWERS TO PROBLEMS 529

(b) Lysine 2.2.3.

12 pK~ 3 ,...._. 2 NH~ 10 I pK =2.18 W a, 8 pH 6 NH ~ predominates at pH 6 4

I pK' = 8.95 W a 2 H N - CH -(CH ) - CH-coo- 3 2 2 4 I NH2 Moles OH- added per mole amino acid titrated

I pK ' = 10.53 (a) The pK~ values are approximately 1.5, 8.5, and W a3 11. The amino acid appears to be cysteine. (b), (c)

H 2 N -CH2 -(CH2)4 - CH -coo- Marked on the figure. l 2.24. Plot c. NH2

predominates at pH 12 CHAPTER 3

2.11. The peptide is cyclic or has a blocked (for example, acetylated) N-terminus. 3.1. 2.12. Glycine does not exist in either an L- or a o-config• uration because it does not possess a chiral center. 2.13. (a) + 1; (b) - 3; pi = 9. 2.14. [+H 3N-CH2 -COO- ]/[H2 N-CH2 -COOH] A B 1.82 X 107 . 2. I 5. (a) pH 12.0; (b) pH 1 3.0. 2.16. MW = 146. t 2.17. A: Asp-Phe; B: Gly-Cys; C: Tyr-Lys. 2.1 fl. Within :±: 1 pH unit of the pK~ values, that is, in the range of pH 1.34- 3.34 and pH 8.69- 10.69. The buffers have maximum capacity at the pK~ values (pH 2.34 and 9.69). 2.19. pH7. 2.20. Aliphatic: a, e, f; aromatic: b, c, d; ac idic: a; basic: e; polar: a, c, d, e; nonpolar: b, f. 2.21. MW = 50,000. 2.22. 6.0 em. pH _.

Protein A is more acidic and has a lower pl. To sep• arate the proteins, lower the pH to piA' at w hich point protein A precipitates. Centrifuge the solution. Protein A forms a pellet, and protein B remains in the supernatant. 3.2. MW = 27,500. 3.3. Lysozyme. 530 ANSWERS TO PROBLEMS

(a) 3; (b) 3; (c) 2, 3, 4. The denatured protein has a more open structure and may have a larger number of functional groups ac• 0 cessible to titration. The pK~ of a group may differ in Polyglwamlo ocid (a) and (b) because the group may be located in dif• 1 ferent electronic environments. MW = 20,000. t 24 H+. Gly-His-Pro-Arg. -70 MW = 50,000. MW = 200,000. Asp-Met-Asp-Met-His-Giy.

Cys-Aia-Pro I Phe-Cys-Leu-Asp -140 L------0 7 14 Since two moles of mercaptoethanol are required to break one mole of disulfide bond, there must be pH..,. three disulfide bonds per molecule of protein. The protein is an oligomer, composed of three identical 5 turns. subunits (MW = 20,000), joined either in series, 4 forms: with one and two disulfide bonds between two sub• a-a-13-13 units, or in a closed triangular arrangement, with one a-13-a-13 disulfide bond at each side. a-13-13-a Ser-Arg-Tyr-Giu-Cys. 13-a-a-13 (a) At pH 7.0, the 13-carboxyl group of aspartic acid Dialyze the protein solution against water, and then is ionized and the imidazole group of is un• add enough solid (NH4 )2 SO 4 (to concentration c1) to charged. The same holds for pH 11.0 so that the in• precipitate protein A. Collect protein A by centrifu• teractions remain unchanged. (b) At pH 3.0, the 13- gation. Protein B remains in the supernatant. Add carboxyl group of aspartic acid still carries a (NH4 )2 SO 4 (to concentration c2), and collect the negative charge, but the imidazole group now car• precipitated protein B by centrifugation. ries a positive charge. Consequently, the attraction between the two groups increases. (c) Increasing the distance between the groups decreases their elec• A B trostatic interaction. 4.88 X 10 8 years. t 4 subunits. (a) 20; (b) 8. (a) No hydrolysis with trypsin. (b) Three cleavages with chymotrypsin:

l pyroGiu-Giy-Pro-Trp-Leu-Giu-Giu-Giu-Giu- 1 l Glu-Aia-Tyr-Giy-Trp-Met-Asp-Phe-(NH2) I 503

3.20. Leu-G ly-Leu-Asp-H is-Tyr-G ly-H is-Phe. 3.21. 50 turns; 27 nm. 3.26. (a) 5.33 X 109 amino acids; (b) 164 peptide bonds per second. 3.27. Leu-His-Arg-Aia-Met-Giu-Ser-Lys-Asp-Met-Lys-Phe• Giy-Met-Lys. ANSWERS TO PROBLEMS 531

CHAPTER 4 hibitor in this reaction. Since competitive inhibition can be overcome by greatly increasing the 4.1. Ordinary enzyme: 15-fold; allosteric enzyme: 2- concentration, the methanol inhibition is effective• fold. ly eliminated by administering large doses of 4.2. pH 5.13. ethanol. 4.3. (a) 80%; (b) 2-fold. 4.24. ES and EP are stable complexes, and X is an inter• 4.4. QlO = 1.77. mediate in the conversion of StoP.(*) Designates an 4.5. 54.6. activated complex. 4.6. (a) [El = [E1] + [ES] + [Ell (b) [El = [E1] + [ES] + [Ell + [ESil (c) [El = [E11 + [ESJ + [ESI] 4.7. Enzyme B. 4.8. Km/(51 = 0.5. 4.9. Ala-Giy is the better substrate because it leads to a smaller Km. The inhibitor having the smaller Ki is the stronger inhibitor. >- e> 4.1 = ][PJ - = Q) 0. d[ESJ/dt k1 [E1][S] + k_ 2 [E1 (k_, + k2)[ES1 0. c 4.11. (a) 45°C; (b) 60°C; (c) 70°C. w 4.12. According to the concerted model, increased con• centrations of competitive inhibitors cause a shift of the equilibrium between the Rand T enzyme forms identical to that produced by increased substrate concentrations. In both instances, large numbers of substrate binding sites are generated. Provided that the inhibitor concentration is low, some of the addi• Reaction coordinate _. tional binding sites can bind substrate, thereby ac• celerating the reaction. 4.25. 75%. 4.13. [51 = 1 .0 X 1o-2 M. 4.2 6. 3.3 enzyme units. 4.14. Km = 5.0 X 1o-3M. 4.27. Vmax =50 nmol ml-1 min-1 ; Km = 6.0 X 10-2 M. 4.15. The second reaction has a greater energy of activa- 4.28. tion. 4.16. (a) Concentrate the solution surrounding the dialysis 1/[S] 1/v 1/v bag by lyophilization (freeze-drying), and add some (normal) (inhibiled) of it back to the enzyme solution. Restoration of en• (mM) (mmol- 1 ml min) (mmol- 1 ml min) zymatic activity indicates loss of an essential cofac• 0.667 5.99 8.70 tor. (b) Dialyze the inactive enzyme solution against 0.500 4.90 6.99 a solution containing high concentrations of sus• pected cofactors . Increasing enzymatic activity as 0.400 4.31 5.99 dialysis proceeds indicates involvement of a dialyz• 0.200 3.20 4.00 able . (c) Determine the optical rotation (he• 0.100 2.60 3.00 lical content) of the active and inactive enzymes. Identical optical activity indicates that the molecule A plot of 1/v versus 1/ [S] shows that the inhibitor is has not unfolded . a competitive inhibitor. 4.17. MW = 25,000. 4.18. Ki = 5.3 X 10-5 . 4.19. A is a precursor of B. C exerts feedback inhibition. Uninhibiled Inhibiled 4.20. The line has a negative slope, - (1/Km), an intercept K., 2.94 mM 5.00 mM on the v/[Sl ordinate of Vma.fKm, and an intercept 1 1 v ma.K 2.00 mmolml- 2.00 mmol ml- max· on the v abscissa of v min- 1 min - 1 4.21 . [1] = 1.0 X 10- 1M. 4.22. The enzyme- substrate complex is more stable than the isolated enzyme and substrate; substrate binding 4.29. IAAcombines with the SH group of cysteine; DFP re• stabilizes the enzyme. acts with the OH group of . Inhibition with ei• 4.2 3. Ethanol is the normal substrate of alcohol dehydro• ther IAA or DFP indicates that both cysteine and ser• genase, and methanol acts as a competitive in- ine are present at the enzyme's . The two 532 ANSWERS TO PROBLEMS

amino acids must be located near each other since 19 different (the two disaccharides once an inhibitor binds to either one, it blocks the marked with an asterisk are identical): second inhibitor from binding to the other amino acid. a-o-glucosyl- 13-o-glucosyl- The rate increases exponentially rather than lin• a-o-glucose 13-o-glucose early because the preparation contains trypsino• (1 -+ 1) (1 -+ 1) gen that is activated to trypsin in an autocatalytic (1 -+ 2) (1 -+ 2) manner. (1 -+ 3) (1 -+ 3) Muscle tissue labeling first increases as labeled (1 -+ 4) (1 -+ 4) amino acids become incorporated into newly made (1 -+ 6) (1 -+ 6) proteins. After reaching a maximum level, the extent a-o-glucosyl- 13-o-glucosyl- of tissue label begins to decrease as proteins degrade 13-o-glucose a-o-glucose (1 -+ 1)* and their amino acids enter . (1-+1)* 80.0%; 1500-fold. (1 -+ 2) (1 -+ 2) (1 -+ 3) (1 -+ 3) (1 -+ 4) (1 -+ 4) (1 -+ 6) (1 -+ 6)

MW = 6480. Every amylose chain contains only one reducing Two molecules of 2,3-dimethylglucose, four mole• end. This represents a very small fraction of reduc• cules of 2,3,6-trimethylglucose, one molecule of ing glucose residues relative to the total number of 1,2,3,6-tetramethylglucose, and three molecules of glucose residues present. When amylose solutions 2,3,4,6-tetramethylglucose. are treated with To liens' and Benedict's reagents, the Initial: +37.4°; final +52.7°. extent of reaction is so small as to be undetectable. (a) Three: reducing end, nonreducing end, and in• 75,585. ternal residues linked via two a(1 ~ 4) glycosidic 10. bonds; (b) five: reducing ends, nonreducing ends, 10%. and internal residues linked via two a(1 ~ 4) glyco• 1: C; 2: D,E; 3: B; 4: D; 5: A; 6: B. sidic bonds or two a(1 ~ 4) and one a(1 ~ 6) gly• cosidic bonds or one a(1 ~ 4) and one a(1 ~ 6) gly• A cosidic bonds. 21.6 g. -11°. ,f-----0 Because the outer membrane acts as a barrier for lysozyme, thereby preventing the enzyme from di• 0 gesting the cell wall. 37% (a); 63% (j3). H OH H OH B (a) (b) (c) 1CHO CHO CHO I I I A------0 2 A----0 H- C-NH-COCH H-C-OH H-C-OH H H I 3 I I H H0-3C-H HO-C-H H-C-H HO-~-H HO-~-H HO-~-H H-~-OH H-~-OH H-~-OH H OH H H I I I c 6CH 20H COOH CHpH

0 5.9. The repeating unit in starch (maltose) consists of two HOH 2QCH glucose residues linked a(1 ~ 4); the repeating unit H H ~0 2 in cellulose (cellobiose) consists of two glucose H HOH 2C residues linked j3(1 ~ 4). 5.10. (a) 4; (b) 3; (c) 5. OH OH OH H ANSWERS TO PROBLEMS 533

Growing cells actively synthesize cell walls in a The following pentasaccharides all yield the same process that includes cross-linking peptidoglycan methylated products as those shown in Figure 5.13: strands. Penicillin inhibits the enzyme that catalyzes this cross-linking and prevents cell wall formation. A Once the cross-links have formed, the cell wall is no longer susceptible to penicillin action. (a) 4; (b) 2; (c) 5. (a) Hydrolyze three consecutive glycosidic bonds at B one end of the amylose chain to produce four frag• ments containing 1, 1, 1, and 97 residues. Average chain length: 25 residues. (b) Hydrolyze three gly• cosidic bonds to produce four fragments containing 25 residues each. Average chain length: 25 residues. c Reduction converts the optically active o-galactose to a meso compound. a-o-Giucopyranosyl-(1 --+ 1 )-a-o-glucopyranoside.

D

E H OH H OH

MW = 180,000. F

G 0~ NH- C- CH -CH- COO- II 2 I H NH 0 NH3 (Figure 5.13) I C=O Because substituents linked via equatorial bonds I generally have greater stability than those linked via CH3 axial bonds, the preponderance of equatorial bonds in cellulose contributes to the great stability of this substance. 534 ANSWERS TO PROBLEMS

27 triacylglycerols (18 optically active):

6 of FA , FA , and FA 1.17 g/ml. 1 2 3 3 of 2FA , FA (a) 0 1 2 II 3 of 2FA1, FA3 CH20-C-(CH2)14 -CH3 3 of 2FA2, FA1 3 of 2FA2, FA3 I ~ 3 of 2FA 31 FA1 CHO-C-(CH2)14 -CH3 + 3KOH - 3 of 2FA3, FA2 CH20H 1 of 3FA1 3CH3-(CH2)14 -COo- K+ + tHOH 1 of 3FA2 I 1 of 3FA3 CH20H Lecithin, ceramide, cephalin, sphingomyelin, phos• phatidic acid, and ganglioside GM2. 1 72 mg of KOH/g of fat. 269 g of iodine/1 00 g of fat. ; phosphatidic acid; sphingosine. An inverted micelle could form. 3.3 mi. At pH 12, phosphatidyl serine, phosphatidyl ethanol• amine, and phosphatidyl choline have net charges of-2, -1, and 0, respectively, and can be fully sep• arated. At that pH, phosphatidyl serine and phos• phatidyl ethanolamine move toward the anode, and CH20H phosphatidyl choline remains at the origin. A diacylglycerol. lI HO-C-(CH2)7CH=CH(CH2)7CH3 ? Six double bonds. Because of the presence of significant amounts of un• CH20H saturated fatty acids, Mb-11 has a less regular structure (c) and is more sensitive to disruption by an increase in temperature. SDS is the compound of choice to dis• 0 II rupt either membrane. SDS forms hydrophobic inter• o c~-o-c~ actions with membrane lipids, thereby breaking simi• II I VVVVVVV\1\ C-0-C-H 0 lar interactions occurring within the bilayer. I II Polar amino acids, because they constitute a site on C~O-P-0- a polar membrane surface and function in the bind• 1 o- ing of polar ATP. + 0.058M. The monolayer area produced by the lipids from one HO -o-~ C~- CH- COO- - I red blood cell is [0.890 X 1012 !J.m 2]/(4.74 x 109 ) Nf-l:i = 188 !J.m2. Since the cell's surface area is only 100 !J.m2, the cell's lipids cover that area 188/100 = 2 times. In other words, they form a bilayer. 2.8 X 106 molecules.

0 The emulsified margarine is a better source of un• i saturated fatty acids because hydrogenation con• 0 C~O-C/VVVVv'\1 II I verts unsaturated to saturated fatty acids. V\1\/VVVV\/\ C- 0-CH 0 6.19. Fat-soluble accumulate in lipid tissues I II C~O- P-0-o-~ C~-CH-C00- whereas water-soluble vitamins are readily excreted. 1 - I 6,20. 21. o- NI-l;; 6.21. (a) 1; (b) 1; (c) 2. 6.22. As bile salts are amphipathic molecules, they are ex• pected to form micelles, monolayers, and bilayers. ANSWERS TO PROBLEMS 535

Denaturation converts dsDNA from a long linear double helix to two globular random coils. The The mutated DNA could hypothetically be repaired change is accompanied by decreases in both vis• by: cosity and optical rotation.

(a) Excising the mutated nucleotide, with or without adjacent , by means of a nuclease. A nu• clease can catalyze the making of two cuts in the damaged strand, one on either side of the mutated nucleotide. (b) Filling the gap by means of DNA polymerase. DNA c: ~o polymerase can catalyze synthesis of a DNA fragment, 0:;:::: ~.s ·- 0 complementary and antiparallel to the intact strand. "'~ uca0- (c) Linking the newly synthesized fragment to there•

maining sections of the damaged strand by means of >a0 a third enzyme.

[A] = 30 mol %. 2.2 X 10-3 micromoles. 3'-TACGGACTCATTG-5'.

(a) Cleavage at A; (b) cleavage at T1; (c) random cleavage. Temperature -. A A A A L t L L pC-G-U-A-c-G-A-U-G-A-G-U-QH 12.5%. t t t t A-DNA: 31.2 nm and 10.9 turns; B-DNA: 40.8 nm T1 and 12.0 turns; Z-DNA: 44.4 nm and 10.0 turns. pK~ = 2.12. Minimum: 200; maximum: 300. No, some DNA is repetitive and some is not ex• OH 0 OH 0 OH pressed. "~"· "~"· The similarity of mitochondrial DNA to bacterial HO~N':)"· HO~~ fy· O~N) HO~)"'"r"· oV DNA-a circular dsDNA of comparable length• H H supports the endosymbiotic theory. According to this theory, mitochondria have evolved from primitive prokaryotes that took up a symbiotic relationship Template: 3' A-T-A-G-C-T-G-C-A-G-T-G-A 5' with early eukaryotic cells. Primer: 5' T-A-T-C-G-A-C-G-T-C-A-C-T 3' Since [G] does not equal [C], and [A] does not equal Composition in mol%: [A] = [ll = 26.9; [C] = [G] = 23.1. [T], the DNA cannot be double-stranded; it must be Thermophiles might be expected to contain larger single-stranded. amounts of G and C than mesophiles. Because G [C] = 14.7 mol%; [T] = 31.8 mol%. and Care linked via three hydrogen bonds, while A 550. and Tare linked via only two, a DNA with a higher [A+ TJ = 73.9 mol%. (G + C) content has a larger number of hydrogen 5' A-C-G-G-C-C-G-T 3' bonds per unit length of double-stranded DNA. The 3' T-G-C-C-G-G-C-A 5' more extensive the hydrogen bonding between the strands (a) -4; (b) -5; (c) -4; (d) 0. is, the greater the thermal stability of the DNA and Mass of a base pair: 1.11 X 1o- 21 g; mass of the the higher its melting temperature (Tm). Increased genome: 6.45 X 10-12 g or 6.45 pg (picograms). thermal stability of DNA may be one of the factors allowing thermophiles to grow at elevated tempera• tures. 536 ANSWERS TO PROBLEMS

The resulting extensive dehydration leads to an un• quenchable thirst. G A c T 1100 kcal for men and 900 kcal for women. 15

14 14 13

12 12 Because there can only be one set of actual condi• 11 tions, regardless of the reference state used; concen• 10 trations, pH, and temperature have fixed values for any given set of conditions. (a) The free energy change for a complex reaction se• quence occurring in vivo must be identical to the free energy change of the simpler in vitro reaction, provided that the initial reactants and the final prod• ucts are the same in both cases. (b) The free energy 5'- [C G TAG] CTG ACTAC G T-3' change of an enzyme-catalyzed reaction is identical to the free energy change of an uncatalyzed reaction 2%. having the same reactants and products. 5' A-C-A-G-G-T-C-C-A-T 3' Inhibitor B. The stronger the inhibitor, the greater is 2 3 4 5 6 7 8 9 10 the equilibrium constant, 1\,q = [EI]/([E][I]), that de• scribes the binding of inhibitor to enzyme. Since .leo = - RT In K~q' inhibitor B has the largest K~q· ile' = -47.6 kJ mol- 1 • .leo= 13.7 kJ mol- 1 • Because control of the committed step determines .leo' (J mol- 1) whether or not the remaining steps of the pathway (2) 2ADP .,t ATP + AMP +2,030 proceed. (3) Glucose + ATP .,o glucose In 150 ml: 1.35 X 1022 H+; in 1000 ml: 9.03 X 1022 6-phosphate + ADP + H+ -19,100 H+. (1) Glucose 6-phosphate .,o glucose A, Matrix; B, outer membrane; C, inner membrane. 1-phosphate +5,706 A is contaminated by 5% of B and 10% of C; B is Glucose + ADP-> glucose 1- contaminated by 7% of A and 11% of C; C is conta• phosphate + AMP + H+ -11 ,364 minated by 8% of A and 13% of B. (overall reaction) 1.08 mg/h. Overall ilCO' = -11.4 kJ mol- 1 ; X~K~G~T. overall Kbio = 98.1. Because newly synthesized polypeptide chains K:q,overall = 1.0 X 102. rapidly dissociate from , exposure to a ra• a and c. dioactive label must be brief so that a significant K:q = 2.59 X w-3. fraction of the label will tag polypeptide chains still ATP4 - + glucose-> AMP-glucose- + PPf• attached to ribosomes. In organisms grown for pro• AMP-glucose- + Pf--> glucose 6-phosphate2 - + longed times in the presence of a radioactive label, AMP 2 - + H+

all newly synthesized proteins will become labeled. Glucose + ATP4 - + Pf--> glucose 6-phosphate2 - + After the proteins dissociate from the ribosomes, AMP2 - + pp~- + H+ their label will spread to other areas of protein me• AG~verall = -43.2 kJ mol- 1• tabolism, resulting in label distribution throughout ile' = -14.7 kJ mol-1 . the cell. Kbio = 3.30 X 10-8 . 8.8. v~s~o~x. 9.11. []/[phosphocreatine] = 3.03 X 107 . 8.9. 3000 mi. 9.14. Mechanism b. ATP synthesis via this mechanism re• 8.10. The rat receiving the 35S-Iabeled compound. quires the more or less simultaneous collision of four 8.11. 13.6 years. entities at each step: ADP, P;, H+, and A, B, or C. By 8.12. Damage to the pituitary results in a drastic decrease contrast, ATP synthesis via mechanism a requires the of vasopressin secretion. Because of the deficiency more or less simultaneous collision of 10 entities: of this hormone, water is not reabsorbed by the kid• 3ADP, 3P;, 3H+, and A. Such multimolecular colli• neys and is excreted in large amounts as dilute . sions are chemically highly unlikely. ANSWERS TO PROBLEMS 537

'J, IS, No, AC cannot be equal to AH. Since AS> 0, the creases the conversion of pyruvate to oxaloacetate term TAS has a finite value and is not equal to zero. and decreases its conversion to acetyl coenzyme A. Accordingly, AH > AC. As in (a), lower levels of acetyl coenzyme A would q if (a) K~q,overall = ([B}[X}[Y])/[A]; K~q, = ([B}[C])/[A]; decrease the activities of the cycle and K~qz = ([X}[Y])/[C]. Thus, K~q,K~q 2 = ([B}[X}[Y])/[A] = the electron transport system. K~q,overall' Muscle phosphorylase leads to energy production (b) [C] = (K~q, [A])/[B] = ([X] [Y])/K~q 2 • Thus, via ; phosphorylase leads to synthe• K~q,K~q 2 = ([B}[X}[Y])/[A] = K~q,overa11· sis of blood . When glycogen is pH 0.76. needed for energy production, such energy must be '1. 111. Yes, the could grow. Since AC' for the con• generated rapidly, hence the large maximum ve• version of citrate to isocitrate is negative, citrate can locity of the muscle enzyme. Restoring the level of serve as a nutrient for the organism. blood sugar, on the other hand, can be carried out 'J I 'l 5.08 moles of ATP. at a slower rate, hence the smaller maximum ve• ll .•!\1 (a) pH= 2.00; (b) K~ = 1.11 X 10-3 ; (c) pK~ = 2.96; locity of the liver enzyme. (d) AGo = 16.9 kJ mol- 1 • Administering ethanol leads to production of [lsocitrate]/[citrate] = 6.70 X 10-2 . NADH and acetaldehyde via the alcohol dehydro• 49.9 kg of ATP; 73.4% of body weight. genase reaction. The NADH can then be used by 5.70 kj mol- 1 . the same enzyme to reduce toxic Kbio = 1.99. back to less harmful methanol. The acetaldehyde AG' = -73.3 kj mol- 1 . formed from ethanol can be metabolized further to 'J.2b. (a) -2884 kj mol-1 ; (b)+ 12.1 kj mol- 1 ; (c) + 2.5 kj acetate. 1 1 mol- ; (d) +13.8 kj mol- . ll. ll. 0.55M glucose. 0. I '. (a) C(3), the methyl-group carbon; (b) C(1 ), the carboxyl-group carbon; (c) C(1 ); (d) C(3). CHAPTFR IU. l. a, c. (a) A 7 -carbon aldose and an 11-carbon 2-ketose; Strenuous exercise exacerbates the effect of alco• (b) an 8-carbon aldose and a 1 0-carbon 2-ketose. hol on . Exercise lowers the levels 30 molecules of glucose. of cellular ATP and NADH and raises that of NAD+. 6ATP. Increased concentrations of NAD+ stimulate the al• A rate-determining step is the slowest step in a re• cohol reaction, the key to gluco• action sequence (smallest rate constant). Rate con• neogenesis inhibition by alcohol. Additionally, ex• stants are unaffected by concentration changes of ercise requires enhanced catabolism reactants and products. However, the rate of a for energy production and leads to an even greater chemical reaction does depend on reactant con• lowering of the blood sugar level than that pro• centration. When glycogen breakdown occurs, the duced by alcohol ingestion alone. glucose level rises and the rate of the hexokinase (a) ACO' = -61.9 kJ mol-1 ; (b) ACO' +13.8 kj reaction increases, leading to more extensive gly• mol-1 . colysis and ATP production. (a) 2 ATP; (b) 2 ATP; (c) 2 ATP; (d) 1 ATP. A rate-determining step is the slowest step in a re• 4ATP. action sequence (smallest rate constant); the se• 3.7 X 1021 molecules of ATP. quence cannot proceed faster than the rate-deter• '· A high Km means that pyruvate is not a particular• mining step. Hexokinase controls the overall rate ly good substrate for the LDH isozyme; a relatively of glycolysis by controlling the concentration of high [S] is required to attain Y2 Vmax· A low kcat also glucose 6-phosphate. A committed step is gener• shows that conversion of pyruvate to lactate does ally a highly exergonic and essentially irreversible not proceed to a great extent. step in a reaction sequence; its occurrence en• In [1 ,3-BPG]/[3PG] = 3.0 X 10-3 • sures that all subsequent steps take place. Phos• 10.8. Activation in (a) and (c), inhibition in (b). Activation phofructokinase catalyzes the formation of fruc• of LDH increases the conversion of pyruvate to lac• tose 1 ,6-bisphosphate, a compound whose sole tate and decreases its conversion to acetyl coen• metabolic role is to serve as a glycolytic interme• zyme A. Lower levels of acetyl coenzyme A de• diate and to ensure that the remaining steps take crease the activities of the and the place. electron transport system. Inhibition of cytochrome 0 l

Because supply ofthe oxidized coenzyme is limit• AG0 ' = -868.4 kJ mol- 1 • ed. NAD+ must be regenerated from NADH so that (a) NADH is a suitable inhibitor because it con• subsequent substrate molecules can be oxidized. stitutes one of the main products of the citric acid cycle. ATP is likewise suitable because the NADH formed enters the electron transport system where it leads to production of ATP. (b) Yes, it is advan• tageous. Inhibition at the early stages of a reac• tion sequence automatically inhibits all of the remaining stages and eliminates unnecessary re• The ratios of NADH/NAD+ and ATP/ADP are high actions. during sleep and low during exercise. Adding the dicarboxylic acids leads to an in• (a) crease in the concentrations of all citric acid cy• cle intermediates and thereby stimulates the cycle's activity, resulting in increased C02 pro• duction. (2) No. The material balance of the cycle is such (1) that two carbons are added in the form of the acetyl group of acetyl CoA and two carbons are released in the form of C02 • No net retention of carbon atoms occurs, and hence no net synthesis of cycle intermediates is possible. The coenzyme A portion of acetyl CoA is released as coenzyme A. Since a high [AMP) indicates a low [ATP], ATP synthesis is needed. By stimulating the complex, AMP leads to produc• tion of acetyl CoA from pyruvate. The acetyl CoA enters the citric acid cycle, and the NADH formed leads to ATP production via the electron transport 1/[S] --+- system. First turn: 0; second turn: one-half ofthe original in• tensity; third turn: one-quarter of the original inten• (b) Yes, the inhibition can be decreased. Malonate sity. acts as a competitive inhibitor of succinate, the 1/4. substrate of . Competi• Competitive inhibition; a small Kr tive inhibition can be overcome by increasing the (a) 11 molecules of ATP per molecule of acetyl CoA; substrate concentration. Adding oxaloacetate (b) 2.5 molecules of ATP per molecule of acetyl leads to an increase in the concentrations of all cit• CoA. ric acid cycle intermediates, of which succinate is [lsocitrate)/[citrate] = 0.133. one. Feedback inhibition. Citric acid cycle: AG0 ' = -33.8 kJ mol-1 • (a) Citrate+ 2NAD+ + GDP + P; +FAD+ H20 45.3%. L OneATP. malate+ 2NADH + 2C02 + GTP + FADH2 Their concentrations as cycle intermediates will de• (b) Acetyl CoA + oxaloacetate + H20 -t citrate crease. + CoA-SH + H+ Yes, labeled C02 is released at step 4, the a-keto• (c) lsocitrate + 2NAD+ + GDP + P; -t succinate glutarate dehydrogenase complex reaction. + 2NADH + 2C02 + GTP Correct. Succinate, one of the intermediates of the citric acid cycle, is synthesized from acetyl CoA by Glyoxylate cycle: the glyoxylate cycle. (a) Citrate + H20 + acetyl CoA-t succinate + 11.22. Yes. Some malate would be diverted from the cit• CoA-SH + H+ +malate ric acid cycle and used for pyruvate synthesis. In• (b) Acetyl CoA + oxaloacetate + H20 -t citrate creasing amounts of pyruvate yield added + CoA-SH + H+ amounts of acetyl CoA for processing by the cy• (c) lsocitrate -t succinate + glyoxylate cle but do not affect the levels of the cycle's in• 11.4. [lsocitrate)/[citrate) = 0.133. termediates. ANSWERS TO PROBLEMS 539

1!.23.

CH3 CH3 I I c=c/ +/ - CH2- N I ~ c - s I CH3 - C - OH I H

Vitamin 81 constitutes the business end of TPP. serves for ATP synthesis. Some energy becomes dis• 11.24. Because the citric acid cycle is obligatorily linked sipated in the form of heat, thereby helping to to the electron transport system via the NADH and maintain body temperature. Diet drinks lack sugar FADH2 produced by the cycle. The coenzymes are and cannot provide heat in this fashion. oxidized by the electron transport system, and this 12.11. 0.5. process is absolutely dependent on the presence of 12. 12. Cells at rest have high levels of ATP and NADH and oxygen. a low level of ADP. Accordingly, such cells have high energy charges. Actively metabolizing cells, on the other hand, have low levels of ATP and CHAPTER 12 NADH and a high level of ADP. These cells have low energy charges. %. 12.1. 2,4-Dinitrophenol is an uncoupler of oxidative 12.13. 41 phosphorylation; it prevents ATP synthesis but per• 12.14. (a) 37%; (b) 80%; (c) 28%. mits electron transport to proceed. Low concentra• 12.15. Succinate + ~0 2 ~fumarate + H2 0. tions of 2,4-dinitrophenol decrease ATP synthesis. 12.16. (a) 72 ATP; (b) 74 ATP. Accordingly, the level of electron transport activity 12.17. Maximum number: 7; likely number: 2. must increase so that ATP demands of the cell can 12. 18. 2ATP. be met. Oxygen consumption increases, and the 12.19. (a) Yes; (b) no; (c) no. P/0 ratio decreases. 12.20. The£"' of NAD+ is - 0 .32 V, and that of succinate 12.2. 6.8xl0- 30/o . is +0.03 V. In order for succinate dehydrogenase's 12.3. Succinate2 - + de hydroascorbic acid,. fumarate2 - coenzyme to oxidize succinate to fumarate, succi• + ascorbic acid; /)P' = +0.03 V, !"lC0 ' = -5.8 kJ nate must be a stronger reducing agent (have a mol- 1, and Kbio = 10.3. smaller reduction potential) than the coenzyme. 12.4. /lC0 '= - 0.074 kj. This relationship holds for succinate and FAD but 12.5. (a) Ascorbic acid + fumarate2 - ~ dehydroascor• not for succinate and NAD+. bic acid + succinate2 - . (b) !lf' = + 0.116 V; !lC' 12.11. 2,4-Dinitrophenol is an uncoupler of oxidative = - 22.4kjmol- 1 . phosphorylation; it prevents ATP synthesis but per• 12.6. [Succinate]/[fumarate) = 40. mits electron transport to proceed. (a) Administer• 12.7. [lactate]/[pyruvate] = 4.6 X 1o- 7 . ing 2,4-dinitrophenol leads to decreased ATP syn• 12.8. The nitrites administered oxidize Fe2+ of hemoglo• thesis. Hence, electron transport activity and bin to Fe3+, thereby forming methemoglobin: respiration must increase so that ATP demands of Hb(Fe2+) ~ Hb(Fe3 +). Methemoglobin competes the cell can be met. This requires degradation of with cytochrome oxidase for cyanide. The compe• large amounts of additional metabolic fuels. Under tition is useful for treating cyanide poisoning be• dietary restrictions, additional metabolic fuels (car• ca use the amount of Hb(Fe3+) that can be formed bohydrates and lipids) come from storage tissues. without impairing oxygen transport greatly exceeds The breakdown of these tissues is expected to re• the amount of poisoned cytochrome oxidase that sult in weight loss. (b) Some deaths occurred be• can be tolerated. cause so little ATP was synthesized that not enough 12.9. [Succinate]/[fumarate] = 2.2 X 1o- 11 . was available to provide the ener gy required for 12.1 0. Soft drinks contain sugar that is catabolized via gly• maintenance of a living state. Also, metabolic fuel colysis, the citric acid cycle, and the electron trans• stores in these individuals may have been insuffi• port system to C02 and H2 0. However, not all of cient to provide the nec essary additional metabo• the energy derived from these catabolic processes lites. It is now known that 2,4-dinitrophenol is 540 ANSWERS TO PROBLEMS

highly toxic to humans because it is rapidly ab• drolysis, X = H2 0, and its two parts are Hand OH; sorbed by all routes of administration (swallowing, in phosphorolysis, X = H3 PO 4 (or some other form contact with intact skin, and inhalation) and is not of P;), and its two parts are Hand H2 P04 ; in thiol• rapidly excreted; it has a biological half-life of 5-14 ysis, X = CoA-SH, and its two parts are H and days. (c) In the presence of 2,4-dinitrophenol, there CoA-S. is a pronounced increase in electron transport ac• The enzyme cascade amplifies the original signal tivity, respiration, and general metabolism. Be• so that significant quantities of fatty acids can be cause little ATP is synthesized via oxidative phos• mobilized rapidly, followed by catabolism to yield phorylation, large amounts of heat are dissipated energy. Because fatty acid oxidation yields many during operation of the electron transport system. AlPs, the cascade allows generation of large This accounts for the rise in body temperature. Ex• amounts of energy in a short time. tensive electron transport activity also means that (a) 5 ATP; (b) 8 ATP; (c) 20 ATP. large amounts of water are produced, resulting in (a) 42 ATP; (b) 461 ATP. profuse sweating. (a) 166,000 kJ; (b) 13.7 days; (c) 766 g/day. Electron transport and ATP synthesis are tightly 968 ml of H20. coupled so that both processes are inhibited by 2.27 g of glycogen, or 8.63 g of glycogen and wa- DCCD. Adding 2,4-dinitrophenol uncouples the ter. two processes; electron transport can proceed, but a, b, c, e, g, i, j, and I. ATP synthesis is inhibited. Yes, the diabetic's breath is likely to contain la• Three molecules of ATP per molecule of NADH. beled acetone. The labeled acetyl CoA adminis• 2,6-Dichlorophenol indophenol: between cyto• tered adds to the pool of acetyl CoA. In diabetics, chrome band Fe-S; TMPD: between cytochrome c acetyl CoA pools tend to be large and are metab• and cytochrome a. olized in part by forming ketone bodies, of which (a) 2; (b) 1; (c) 1. acetone is one. 125,000 g; 2500 times. (a) No 14C02 will be produced. (b) No 14C02 will (a) 7 ATP; (b) 37 ATP; (c) 16 ATP. be produced from the last acetyl CoA itself as it (a) 30 ATP; (b) 18 ATP. passes through one turn of the citric acid cycle. However, as this acetyl CoA passes through the cycle, 14C02 will be released because all of the cycle's intermediates have by now become la• beled from previous turns. Labeled water will (a) 12 Acetyl CoA, 11 FADH 2 , and 11 NADH; (b) 9 form in both (a) and (b) since steps 6 and 8 of the acetyl CoA, 9 FADH 2, and 9 NADH. cycle (Figure 11.1 0) yield 3 H-Iabeled FADH 2 and All of the carbons will become labeled. (NADH + H+). Oxidation of these coenzymes via 11 carbons. the electron transport system produces labeled 65 molecules of glucose. water. fj-Oxidation of fatty acids derived from adipose tis• (a) + 2NAD+ + P; + ADP--? pyruvate + sues produces large quantities of FADH 2 and 2NADH + 2H+ + ATP + H20 NADH. When these coenzymes enter the electron (b) Propionyl CoA + HC03 + ATP --? succinyl transport system, metabolic water is produced. CoA + ADP + P; + H+ (a) CH 3 (CH 2 ) 18COO- + 9FAD + 9NAD+ + (c) 2 Acetyl CoA + H2 0 --? acetoacetate + 9H20 + 10CoA-SH4 - + ATP4 - 2CoA-SH + H+ ! The person is better off consuming odd-numbered 10 acetyl CoN- + 9FADH2 + 9NADH + fatty acids because their catabolism generates not 9H+ + AMP2 - + PP; 3 - only acetyl CoA but also propionyl CoA. Catabo•

(b) CH 3 (CH 2 )13Coo- + 6FAD + 6NAD+ + lism of acetyl CoA provides energy. Catabolism of 4 4 6H 20 + 7CoA-SH - + ATP - propionyl CoA produces succinyl CoA and leads to ! an increase in the concentrations of the citric acid CH 3 CH 2CO-S-CoN- + 6 acetyl CoN- + cycle's intermediates. One of these, oxaloacetate, 6FADH2 + 6NADH + 6H+ + AMP2 - can then be used in gluconeogenesis to provide + pp3- some of the carbohydrate lacking in the person's 13.7. All three mec'hanisms involve cleavage of a cova• diet. lent bond by compound X such that one part of X 13.19. By converting the hydrocarbons to carboxylic adds to one cleavage product, and the other part acids and then oxidizing these completely to C02 of X adds to the second cleavage product. In hy- and H2 0. Because an oil spill consists of hydro- ANSWERS TO PROBLEMS 541

carbons, use of such organisms will clean up the Glucose + 2NAD+ + 2~DP + 2P; + 2 aspartate spill. 24 ATP. 2 + 2 oxaloacetate + 2ATP + (a) Cycle 1; (b) cycle 1; (c) cycle 4; (d) cycles 1 and 2NADH + 2H+ 2; (e) cycle 7. IMP synthesis: 3 ATP; UMP synthesis: 2 ATP.

CH 3(CH 2 )6COOH + 1102 ~ 8C02 + 8H20 is caused by abnormal metabolism of uric (b). acid, the end product of catabolism in ~s moles of palmitate/mole of glucose. humans. Because amino acids are precursors of (a) 29 ATP; (b) 6 ATP and 12 (NADPH, H+). Because and , the protein-rich diet NADPH can be converted to NADH, the require• (the meat diet) is more likely to lead to excessive ment of 12 (NADPH, H+) constitutes a potential formation and to the development of loss of 36 ATP (assuming that no shuttle is needed), gout. so that the total effective requirement is 42 (a) Glutamate- + NAD+ (NADP+) + H 2 0 ATP/molecule of myristic acid. l The ATP requirement for myristic acid synthe• a-ketoglutarate2- + NADH (NADPH) + H+ + NH; sis and the ATP yield upon degradation of the fatty (b) Glutamate- + NH! + ATP 4 ---> acid are not identical because an anabolic pathway + ADP3 - + p~- + H+ can never be the exact reverse of the correspond• (c) Phenylalanine + Of + tetrahydrobiopterin ing catabolic pathway. requires more energy than can be derived from fatty acid tyrosine + H20 + quinonoid dihydrobiopterin degradation. The same holds for glucose. Glucose (d) + + H2 0--> ornithine+ + urea synthesis from pyruvate requires 6 ATP equivalents (e) + phosphoribosyl pyrophosphate5 - per glucose, but glucose catabolism to pyruvate l yields only 2 ATP per glucose. adenosine 5'-phosphate2 - + PP(- Malonyl CoA will contain 14C, but this is lost as (f) 5'-triphosphate4 - +reduced 14C02 in step 2 of fatty acid biosynthesis, so that the palmitate produced will not be labeled. l (a) Three D at C(16); (b) two D at each of the fol• deoxycytidine 5' -triphosphate4 - + oxidized

lowing: C(14), C(12), C(l 0), C(8), C(6), C(4), and ribonucleotide reductase + H 2 0 C(2). Yes for , no for iodoacetamide. Reduc• Increased fatty acid mobilization; ; fatty liver. tion of ribonucleotides requires the reduced form of ribonucleotide reductase. Because glutathione acts as an , adding it will stabilize the enzyme in its reduced state and preserve the en• zyme's critical sulfhydryl groups. lodoacetamide is an irreversible inhibitor of the enzyme because (c). it combines covalently with sulfhydryl groups. Although arginine is synthesized in young organ• Adding this compound will inactivate the en• isms, its rate of synthesis is insufficient to meet the zyme. 14 14 nutritional requirements. C02 does not form from [ C]acetyl CoA during (a) 9; (b) 32. the first turn of the citric acid cycle but does form

Glutamate labeled at the a-carbon. during subsequent turns. Because C02 in the form Yes, both amino groups will become labeled. of HC03 constitutes a precursor of carbamoyl [15N]Aianine can give rise to [15N]aspartate by phosphate, labeled (and transamination and to 15NH: by oxidative deami• hence labeled urea) will form as soon as 14C02 is nation. 15NH: can lead to [15 N]carbamoyl phos• produced. phate by one of the fixation reactions. Since one of the amino groups in urea comes from aspartate while the other comes from carbamoyl phosphate, both of urea's amino groups will be• come labeled with 15N. 14.6. o-Phenylalanine + ~0 2 ~phenyl pyruvate+ NH: 14.7. (a) Nitrogenase; (b) nitrate reductase; (c) nitrite re• ductase; (d) carbamoyl phosphate . 14.8. (a) 0; (b) +1; (c) +3; (d) +5; (e) -2; (f) -1; (g) -1. 542 ANSWERS TO PROBLEMS

NH+ 0 mulating methylmalonyl CoA with myelin sheath I 3 II formation. (a) CH -CH-coo- ~ CH -C-coo- 14.11 3 3 -LIB. 15 AlP/molecule of alanine. Alanine Pyruvate q Fasting lowers the level of blood sugar. Subsequent NH+ administration of amino acids results in rapid catab• I 3 olism of glucogenic amino acids. Oxidative deami• (b) -ooc-cH-CH -CH -coo- ~ 2 2 nation of the amino acids produces ammonia, but Glutamate the arginine deficiency prevents adequate conver• 0 sion of ammonia to urea. As a result, the concentra• II tion of ammonia in the blood increases sharply. -ooc-c-CH -CH -coo- 2 2 1-L!O. Because of their extensive nucleic acid breakdown, a-Ketoglutarate afflicted individuals have high levels of uric acid NH+ that can produce kidney stones and gout. Allopuri• I 3 nol, an inhibitor of oxidase, prevents uric (c) -ooc-cH-CH -coo- ~ 2 acid formation. Administering allopurinol decreas• Aspartate es uric acid accumulation and lowers the chance 0 that patients will develop kidney stones or gout. II HGPRT deficiency causes an increase in the con• -ooc-c-CH -coo- 2 centration of PRPP, and high levels of PRPP lead to oxaloacetate excessive production of purines and pyrimidines. NH+ The effect is especially pronounced for purines, - I 3 whose synthesis essentially begins with PRPP. High (d) 0-CH -CH-COO- 2 ~ purine concentrations result in enhanced purine Phenylalanine \_jl catabolism. The resultant accumulation of uric 0 acid can lead to kidney stones and gout. Why HG• PRT deficiency leads to the neurological and be• ;='\ cH -~-coo- havioral symptoms described is not known. ~Phe~ylpyruvate A high-protein diet results in increased production of NH+ urea. Drinking large amounts of water increases the I 3 volume of urine and allows for excretion of the urea (e) Ho-Q9 -CH 2 -~H-coo- ~ from the body in the form of relatively dilute solu• II Tyrosme tions. This puts less strain on the kidneys than would 0 the excretion of more concentrated urea solutions. -I. 2 l. Three energy-rich bonds. Ho-<==)-cH 2 -~-coo- ·L24. Five a-amino acids participate in the : ornithine, citrulline, aspartate, argininosuccinate, and p-Hydroxyphenyl pyruvate arginine. Of these, only aspartate and arginine are The transamination reaction yields pyruvate and used for . glutamate. Inclusion of lactate dehydrogenase re• 1 sults in conversion of pyruvate to lactate, with the concomitant oxidation of NADH to NAD+. Since NADH, but not NAD+, absorbs at 340 nm, progress of the reaction can be followed by measuring the decrease in absorbance at that wavelength. 14.1 Three enzymatic steps convert propionyl CoA to succinyl CoA. The third enzyme in this sequence (methyl malonyl CoA mutase) requires a derivative of B12 as coenzyme. Because of the defi• cient absorption of in pernicious ane• mia, methylmalonyl CoA cannot be metabolized further, and the catabolism of isoleucine, methion• ine, and valine is impaired. Researchers believe that the neurological deterioration associated with the disease results from interference of the accu-

Time- ANSWERS TO PROBLEMS 543

Since aspartame is a derivative of phenylalanine, the Six photons must strike each , and six individual would be better advised to use saccharin. electrons must flow through the Z-scheme. Skin pigmentation results from the presence of mel• 0.5. anins, which are polymeric subtances derived from 26.7%. tyrosine. Since kwashiorkor is a disease of protein (a) -4; (b) -3. deficiency, the lack of tyrosine prevents No, since Rhodopseudomonas viridis uses a cyclic formation. electron flow that involves only one photosystem 117 g/day; 16.4%. at 870 nm.

Amino acid catabolism accounts for the difference. /lG0 ' = -96.5 kj mol-1 • One photon can excite Compared to degradation of carbohydrates, amino one molecule of P870. acid degradation to pyruvate or to intermediates of 1/6. Fractions of 14C in fructose 6-phosphate: 1/3; the citric acid cycle lacks the energy-yielding steps 1/6; 0. of glycolysis. Compared to degradation of lipids, The colors of the accessory pigments are normally amino acid degradation generates much less acetyl masked by that of . When leaves lose CoA. Lastly, while carbohydrate and lipid catabo• their chlorophyll, colors of the accessory pigments lism are exergonic, a good part of protein catabo• become noticeable, accounting for the multiple lism-the urea cycle-is endergonic. colors of fall foliage. The rapid growth characteristic of many cancer 11.9%.

cells requires an above-average supply of nu• I:J.£0' = +0.11 V; /lG0 ' = -21 kj mol- 1 • cleotides. Consequently, such cells are particularly of the C3 leads to a decrease sensitive to inhibitors of purine or in the concentration of C02 • As the concentration biosynthesis. Because viruses also replicate rapid• of C02 falls, becomes more pro• ly, viral systems may be affected similarly by action nounced and lowers the photosynthetic efficiency. of the same inhibitors. Increased photorespiration also results in apprecia•

ble water loss by the plant. By contrast, the C4 plant avoids wasteful photorespiration and suffers mini• mal water loss. No, since the sequence QA ~ QH2 is not involved No, since a free energy change calculated from in the electron transport system of cyclic photo• 0 phosphorylation. !:J.£0' yields I:J.G '. To properly evaluate intracellular reactions, !:J.G' values must be used. Ferricyanide acts as an artificial electron acceptor, accepting electrons from one of the plastoquinones 2H2 0 ~ 4H+ + 4e- + 0 2 (Q) preceding QH (Figure 15.9). QH2 + 2(cyt bf)ox ~ Q + 2(cyt bf)red + 2H+ 2 Since photorespiration decreases the efficiency of photosynthesis, the mouse in (a) will last longer than that in (b). The mouse in (c) will last the longest because C4 carry out photosynthesis with greater efficiency than c3 plants. 2.5 X 1011 tons per year. No. Both involve only visible light; they are unaffected by ultraviolet radiation, which has shorter wavelengths. Also, the two known effects of light on photosynthesis-red drop and the Emerson enhancement effect-require light in the visible range. 2/3= 0.67. : -400-500 nm, violet-blue; phyco• cyanins: -600 nm, yellow; phycoerythrins: -500-600 nm, green-yellow. 15.8. PSI: 3NADP+ + 3H- + 3W ~3NADPH + JH+ (6H+ + 6e-)

Oxygen acts as a competitive inhibitor of the en• PSII: zyme. The apparent Km (02 atmosphere) is greater 12 hv than the true Km (N 2 atmosphere) of the enzyme. Overall: 3NADP+ + 3H 0--> 3NADPH + 2 15.22. !:J.G' = -55.9 kj mol-1 • JH+ + 0 3 15.23. X. = 385 nm. 544 ANSWERS TO PROBLEMS

15.2-l. ilf' = 0.29 V. AGC-(UCU-CUC)n -· · · ·-CUG-G 15.25. ilE' = 2.5 V. Ser (Ser Leu) n Leu I 'i.26. (a) Impossible. Since there is no external electron A-GCU-(CUC-UCU)n - · · · ·-UGG donor, net NADPH production cannot occur. (b) Ala (Leu Ser)n Trp Possible. While the scheme is theoretically plausi• AG-(CUC-UCU)n -· · · ·-UCU-GG ble, it is practically unlikely because it requires a (Leu Ser) n Ser pigment capable of generating a very large poten• 16.6. p = 0.0338 or 3.4%. tial difference of about 2 V. (c) Possible. (d) Possi• 1 6.7. ble.

Base composition Sequence Probability (%) CHAPTE 16 [UUC]: Two U, one ucu 12.8 C; probability = uuc 12.8 16. I . Using the layout of Figure 16.11, you can show that (0.80)(0.80)(0.20) cuu 12.8 the minimum number of different tRNAs required 38.4 to "recognize" the amino acid codons is 32: [CCUJ: Two C, one ccu 3.2 u c A G U; probability = cue 3.2 (0.20)(0.20)(0.80) ucc 3.2 9.6 u 2 2 1 2 UUU: Three U; UUU 51.2 c 2 2 2 2 probability = (0.80)(0.80)(0.80) A 3 2 2 2 51.2

G 2 2 2 2 CCC: Three C; CCC 0.8 probability = (0.20)(0.20)(0.20) I (> .2. In the mitochondrial code, a single codon is used 0.8 for each of the eight four-codon families (Leu, Val, 100.0 Ser, Pro, Thr, Ala, Arg, and Gly). Other changes are

shown in Table 16.4. Using the same layout as in 1 6.8. If the oligonucleotide is translated in the 5' ~ 3' di• the previous problem, you can show that the rection, the product is oligoalanine having threo• minimum number of different tRNAs required to nine as its N-terminus. If translation is in the 3' ~ "recognize" the amino acid codons is 22: 5' direction, the product is oligoproline having threonine as its C-terminus. u c A G l b.9. Translation of the oligonucleotide in theS' ~ 3' di• rection yields the oligopeptide Cys(Leu)n. Because u 2 1 1 2 the leucine sequence increases in length with in• creasing translation time, the data prove that the c 1 1 2 1 polypeptide chain grows from the N- to the C-ter• minus. In. I 0. Lack of base complementarity indicates that the A 2 1 2 1 DNA is single-stranded. Resistance to attack by ei• ther phosphodiesterase I or II indicates that the G 1 1 2 1 DNA has neither a free 5'-0H nor a free 3'-0H. You conclude that the DNA is single-stranded and 16.3. 5'-CTG-3'. circular. 16.-t. 3'-GAC-5'. 16.1 1. N-Thr-Leu-Thr-Asp-Cys-Pro-Arg-C. 16. 5. Three different polypeptide chains form depending 1 6.12. (a) N-Thr-Leu-Asp-Gly-Leu-Pro-C. on the point at which translation of the oligonu• (b) N-Thr-Leu-Thr-Aia-Aia-Leu-C. cleotide begins. The bulk of all three polypeptide 16. I 3. The nature of the peptide product depends on the chains is an alternating sequence of serine and point at which translation of the oligonucleotide leucine residues, but the chains differ in the struc• begins: (a) Polyarginine, polyglutamic acid, and tures of their N- and C-termini: polylysine; (b) polyglutamic acid and polylysine. 16.14. Changing normal to sickle-cell hemoglobin in- ANSWERS TO PROBLEMS 545

volves replacing a (codons GAA and CHAPTER 17 GAG) by valine (codons GUU, GUC, GUA, and GUG). Two codon conversions can be accom• 17 .1. Since A/T-rich segments have fewer hydrogen plished by single base mutations: GAA - GUA bonds than G/C-rich segments, they are easier to and GAG - GUG. unwind; local unwinding of the double helix is a 16.15. 51 bp. prerequisite of replication. 16.16. Four types of intact ribosomes can form: 17.1. DNA replication requires the participation of RNA; it cannot proceed without the aid of RNA primers. "All heavy": Heavy 305 + heavy 50S This suggests that a simpler mechanism involving "All light": Light 30S + light 50S RNA replication evolved first and subsequently Hybrid: Heavy 30S + light 50S changed to the more complex mechanism of DNA Hybrid: Light 30S + heavy 50S replication. 17.3. (a) 5' U-G-A-C-A-G-C -U-A-C -C-U• 16.17. Phenylalanine. G-G-U3' 16.18. 5'-AAUCGUC-3'. (b) 5' T-G-A-C-A-G-C-T-A-C-C-T• 16.19. Isoleucine, threonine, or . G-G-T3' 16.20. In the overlapping code of Problem 16.19, most ~5'U-A-A-C-A-A-C-U-A-C-C-U­ bases are used three times. Each base occurs in A-A-U3'

three different codons and at a different position in 17.4. At t1 : 1000 nucleotides; at t2 : 1818 nucleotides. each: 5'-end, middle, 3'-end. In these cases, a sin• 17.5. 6580 cells. gle base mutation leads to three altered codons. 17.6. and testosterone because they have planar Unless two of the altered codons are synonym structures. codons, the mutation results in three amino acid 17.7. 4000 Okazaki fragments. changes in the corresponding protein. 17.8. 1 .28 years. 16.21. a, b, and c. 17.9. Because DNA is entirely double-stranded whereas 16.22. (a) 5'-CCU-3'; Pro RNA is either single-stranded or only partially dou• 5'-CCC-3'; Pro ble-stranded. The double-stranded structure allows 5'-CCA-3'; Pro for accurate DNA replication through synthesis of (b) 5'-UCA-3'; Ser two complementary and anti parallel strands. It also 5'-UCG-3'; Ser allows for ready DNA repair. (c) 5'-UGU-3'; Cys 17.10. Hybrid DNA (ds): 2/8 or 25%; "all light" DNA (ds): 5'-UGC-3'; Cys 6/8 or 75%; "heavy" single strands: 2/16 or 12.5%; 16.23. a, c, e, and f. "light" single strands: 14/16 or 87.5%. 16.24. mRNA: [U] = 33.3 mol %, [G] = 66.7 mol %; 17 .11. a, d, and f. The 13- and 'Y-phosphate groups of dTIP DNA: [A] = [T] = 16.7 mol %, [C] = [G] = 33.3 are cleaved out in the form of PP; as dTMP is in• mol%. corporated into the growing polynucleotide strand. 16.15. Working backward from , you can es• Hence, only dTTP forms containing labeled ex• tablish the following underlined sequence of single phosphate groups are useful for the assay. point mutations: 17.12. (a)-·.. ···; (b) ...... 17.13. B-DNA: 4.0 X 105 turns; Z-DNA: 3.3 X 105 turns. Leu ---+ Pro ---+ Ala ---+ Gly ---+ Trp 17.14. E. coli: 0.31 s; eukaryotes: 4.7 s. ccu GCU GGU UGG cuu 17.15. 0.12 em/h. cue CCC GCC GGC/-• CUA CCA GCA GGA 17.16. 5.6 X 104 molecules of glucose. 17.1 7. "All heavy" DNA (ds): none; hybrid DNA (ds): 2/16 CUG ---+ CCG ---+ GCG ---+ GGG UUA or 12.5%; "all light" DNA (ds): 14/16 or 87.5%; UUG "heavy" single strands: 2/32 or 6.25%; "light" sin• gle strands: 30/32 or 93.75%. 16.26. 5'-ACT-3'. 17.18. 250 replication forks. 16.27. Five times. 17.19. 5 mg; 2.5%. 16.28. Arginine: [AGGJ; lysine: [AAA]. 17.20. Phenylalanine and leucine. 16.29. 312 base pairs. 17.21. (a) 5.0 X 104 bp/min; (b) 17 iJ.m/min. 17.22. DNA polymerase I and DNA ligase. 17.23. G·C. 17.24. 5' G-A-T-C-A-T-A-T-G-A-T-C 3' 3' C-T-A-G-T-A-T-A-C-T-A-G 5' 17.25. Because of its structural similarity to deoxyribonu- 546 ANSWERS TO PROBLEMS

cleoside triphosphates (the normal substrates of of each protein in the cell is a function of its rate of DNA polymerase), 5-fluorouridine triphosphate is degradation or half-life. likely to act as a competitive inhibitor of the en• Because attenuation requires tight coupling of tran• zyme. scription and translation. This coupling, which oc• Because nuclear DNA controls most cell functions curs in prokaryotes, allows the two processes to whereas mitochondrial DNA is limited to coding proceed simultaneously in the same subcellular for mitochondrial protein synthesis and a few oth• compartment. Coupling of transcription and trans• er proteins. lation is impossible in eukaryotes because the two FAD, FMN, CoA-SH, or NADP+ might function in processes take place in separate subcellular com• the reaction because each of these molecules con• partments. tains an AMP moiety. GTP might substitute for ATP The trp attenuator contains two tryptophan codons, as the two compounds differ only in the nature of but the his attenuator contains seven histidine their purine ring. Lastly, AMP itself might interact codons. Accordingly, a larger number of amino directly with the enzyme. acid molecules are needed to terminate transcrip• 1.733 g/cm3. tion by attenuation for the his operon than for the trp operon. If you add an equal number of amino acid molecules, attenuation of the trp operon will be more pronounced.

5 I -AGC-CAU-UCG-AGG-UGU-UCA-CGU-AAA-3'. (a) An inducer/repressor complex in enzyme in• Ser-His-Ser-Arg-Cys-Ser-Arg-Lys. duction and a co-repressor/repressor complex in For any given section of double-stranded DNA, enzyme repression. The inducer/repressor complex only one of the two strands is transcribed into RNA. prevents the repressor from binding to the operator, (a) 24.0 s; (b) 53.3 s. thereby allowing transcription to proceed during Since the mRNA has a short half-life, the label re• enzyme induction. The co-repressor/repressor maining after a prolonged time (40%) represents complex binds to the operator and turns it off, that taken up by other types of RNA. Accordingly, thereby stopping transcription during enzyme re• the amount taken up by mRNA is approximately pression. (b) A cAMP-CAP complex. This complex 100% - 40% = 60%. All of the mRNA label is lost binds to the promoter, stimulates attachment of within about 5 min (300 s) from the start of the ex• RNA polymerase, and increases transcription of the periment. About half of the amount incorporated lac operon so that lactose is metabolized. into mRNA (30%) is lost within about 180 s from (a) 40; (b) 59; (c) 60. the start of the experiment or within (180 - 60) = 120 s from the time the chase was added. By this s.o4 x 1o 6 . 2.51 X 10-15 g. time, the incorporation into mRNA has dropped by 30% (one-half of the maximum 60%) from 1.89 X 106 . DNA base sequence: 100% to 70%, indicating an approximate half-life of 120 s. 3 I-TAC-TAC-ACC-ACC-TAC-ACC-ACC-5 I Further transcription is inhibited because cordy• 5 I -ATG-ATG-TGG-TGG-ATG-TGG-TGG-3 I DNA base composition: [A] = [TJ = 10/42 = 23.8 cepin 5'-triphosphate lacks the free 3'-0H re• mol%; [C] = (GJ = 11/42 = 26.2 mol%. quired for elongation of the strand. The inhibition Determine the base composition of the 165 rRNA. indicates that the polynucleotide strand grows in RNA transcribed from both strands of DNA must the 5' ~ 3' direction. show base complementarity so that [A] = [U] and The sigma subunit would compete with the [G] = [C]. holoenzyme for binding to the promoter. Because Both terms may be applied to the inducer of the lac the holoenzyme is essential for initiation of tran• operon. The inducer counteracts the repressor; it scription, the sigma subunit would act as a com• functions as an antirepressor. In the absence of in• petitive inhibitor and lead to a decrease in the rate ducer, the operator is blocked and the operon is re- of transcription. pressed. Thus, the inducer acts as a de-repressor. 18.19. The core enzyme by itself must bind more tightly The co-repressor of the trp operon also functions as to the DNA than the core enzyme associated with an anti repressor in the sense that it counteracts the the sigma subunit. Failure of the sigma subunit to repressor, but it does not function as a de-repressor. dissociate from the holoenzyme would make the 18.9.. An incorrect conclusion. The three genes are tran- mutant enzyme less processive, resulting in slower scribed as a unit, and the mRNA is translated as a RNA synthesis. unit, resulting in synthesis of the three gene prod- 18.20. Deletion of the regulatory gene means that the re• ucts in equal numbers. However, the accumulation pressor is not synthesized. Consequently, both lac- ANSWERS TO PROBLEMS 547

tose and tryptophan synthesis would be continu• No energy-rich bonds are expended at this step; ous. In the case of the lac operon, the operator is fiG' =0. not turned off because of the absence of a repres• Both act on the 505 subunit of prokaryotic ribo• sor. In the case of the trp operon, the operator is not somes and block chain elongation. turned off because of the absence of a co-repres• 90 nucleotides per second. sor/repressor complex. 600 energy-rich bonds. Determine the distribution of poly(A) tail lengths in 3600 energy-rich bonds. bulk mRNA. Finding that the tail in all mRNAs has (a) All protein molecules from both experiments a uniform size would argue against both parts of the have the same N-terminal amino acid. (b) In the ab• proposed hypothesis. Finding that the tails have sence of puromycin, all protein molecules have the varying lengths would support part (a). Finding that same C-terminal amino acid. In the presence of the distribution of tail lengths is cut off at a partic• puromycin, partially completed chains of the pro• ular minimal value (5 or some other value) would tein end in various C-terminal amino acids. (c) In support part (b). the presence of puromycin, the average chain [A] = [T] = 20 mol %; [C] = [G] = 30 mol %. length is shorter because polypeptide chains are (a) In the absence of active (3-galactoside perme• prematurely terminated. ase, lactose cannot be transported from the medi• 6.4 kg. um into the cells. Consequently, lactose cannot be (a) 0.990; (b) 0.912. utilized to any significant extent. (b) Lactose uti• Met-Pro-Asp-Phe-Met-Vai-Gin-Leu-Ser-Ser-Giu-Met. lization by the cells will increase. (a) Met-Ser-Asp-Phe-Met-Vai-G I n-Leu-Ser-Ser-G Iu• (a) Synthesis of a protein from an mRNA transcript Met. of genes C, B, and A. (b) No tryptophan synthesis. (b) Met-Pro-Asp-Phe-Met-Vai-G I n-Leu-Ser-Ser• [A]= 25 mol%; [U] = 19 mol%; [C] = [G] = 28 Giu-Met. mol%. (c) Met-Pro-Asp-Phe-Met-Val. mRNA generally has a short half-life or high 0.42 min (f. coli); 8.3 min (rabbit reticulocytes). turnover; it is synthesized and degraded rapidly. Their mechanisms of action involve a that The intact double-stranded segment most likely catalyzes the phosphorylation of initiation factor represents that section of the DNA that binds the eiF-2, thereby blocking translation. Heme-con• RNA polymerase holoenzyme. Binding of the RNA trolled inhibitor is a kinase ofthis type, whereas in• polymerase protects the DNA against by terferons may induce the enzyme. deoxyribonuclease. Determining the base se• (a) and (c). In preparation (b), protein synthesis will quence of the intact segment helps to identify the not be stimulated because eukaryotic initiation re• promoter site of the DNA. quires methionyl-tRNA. In preparation (c), protein synthesis will be stimulated because mitochondri• al initiation also proceeds with N-formylme• thionyl-tRNA. (a) 3'-CAG-5'; (b) 3'-CUG-5'. Codon UGU. The enzyme must have at least three binding sites By inhibiting a specific protein-synthesizing sys• for an incoming amino acid, ATP, and tRNA. In ad• tem with puromycin (for example, reticulocytes dition, the enzyme must bind the two products, synthesizing hemoglobin, or bacterial cells in• aminoacyl adenylate and aminoacyl-tRNA. duced to produce (3-galactosidase) and assaying (a) A, C, B; (b) X, Z, Y. (d) and (e). the peptidyl puromycin formed for protein struc• ture and/or activity. If the protein begins to fold Based on the wobble hypothesis (Table 16.5): Codons: 5'-CGU-3' 5'-CGC-3' 5'-CGA-3' 5'-CGG-3' before its synthesis is complete, the peptidyl Anticodons: 3'-GCA-5' 3'-GCG-5' 3'-GCU-5' 3'-GCC-5' puromycin may possess a portion of the protein's 3'-GCG-5' 3'-GCI-5' 3'-GCI-5' 3'-GCU-5' final three-dimensional structure. Depending on 3' -GCI-5' what section of the molecule has folded, the pep• GC base pairs are linked via three hydrogen bonds; tidy! puromycin may also possess some or all of all others (AU, AI, Ul, Cl, and GU) are linked via the protein's activity. two hydrogen bonds. Accordingly, the underlined 19.3. 0.25 mg. anticodons result in the strongest hydrogen bond• 19.4. (a) 3; (b) 8; (c) 5; (d) 4; (e) 12; (f) 1; (g) 9; (h) 2; (i) 6; ing with their codons. These anticodon-codon (j) 11; (k) 10; (I) 7. pairings release the tRNA more slowly, resulting in 19.5. 18 amino acids. slower rates of protein synthesis. 19.6. 9 amino acids. 19.26. Met-Leu-Asn-Leu-Arg-Tyr-Pro. 19.7. (b). 19.27. Modify the bacterial DNA so that the protein's gene 548 ANSWERS TO PROBLEMS

leads to synthesis of a polypeptide chain enriched Since leucine has six codons, adding it to the in nonpolar amino acids at the N-terminus. If at tripeptide leads to 6 X 8 = 48 possible mRNA se• least 10-15 of the protein's first 30 amino acids are quences. Adding valine (four codons) to the hydrophobic in nature, the N-terminal segment tetrapeptide raises the number of possible se• might function as a signal peptide. In that case, syn• quences to 48 X 4 = 192. thesis of the protein would be completed by mem• You should purify phenylalanyl-tRNA synthetase. It brane-bound ribosomes, and, once synthesized, is thought that an aminoacyl-tRNA synthetase can the protein would be secreted across the cell mem• screen out, by hydrolysis, mischarged amino acids brane. smaller than the correct amino acid. Since glycine There are eight possible mRNA sequences: is the smallest amino acid, glycyl-tRNA synthetase may not possess such a hydrolytic site, and study• CAU ing this enzyme might not be useful. AUG-GCU/\ CAC /CAU AUG-GCC\ CAC ;CAU AUG-GCA\ CAC ;CAU AUG-GCG\ CAC Index

Abiotic reactions, 4 N-Acetylgalactosamine, 124, 132-134 Adapter RNA: see Transfer RNA ABO system, 137 N-Acetylglucosamine, 124, 132-136,268 Adenine, 172 Absorbance, 511 N-Acetylhexosaminidase, 21 OT pK~ values, 173F Absorption N-Acetyllactosamine, 268 Adenosine, 174 oflight, 379-380, 511 N-Acetylmuramic acid, 124, 135-136 , 368F of nutrients, 213-214 N-Acetylneuraminic acid: see Sialic acid Adenosine 5'-diphosphate (ADP), 175 Acanthocytosis, 21 OT Acetylsalicylic acid (aspirin), !50 in energy charge, 307-308 Acceptor RNA: see Transfer RNA Achromic point, 115 in regulation of metabolism, 250, 286F Acceptor site: see Aminoacyl site Acid anhydrides, 228 Adenosine 5 '-monophosphate (AMP), 175 Accessory pigments, 382-383 Acid-base , 90 in energy charge, 307-308 Acetal, 120-121 Acid dissociation constant (K~), 14: see also in regulation of metabolism, 250, 267F,

Acetaldehyde, 206 p K'a 279,364F Acetate thiokinase, 282 Acidification of urine, 23 Adenosine 5' -triphosphate: see ATP Acetic acid Acidosis, 13, 329 S-Adenosylmethionine (SAM), 318-319, free energy of ionization, 222-224 , 282 459 pK~ value, 16T cis-Aconitate, 282 Adenylate cyclase, 258-259, 262F Acetoacetate, 327-329, 328, 354 ACP: see Acyl carrier protein Adenyl ate kinase, 208T, 307 Acetoacetyl-ACP, 333-334 Acquired immunodeficiency syndrome Adenylic acid: see Adenosine 5' -monophos- Acetoacetyl CoA, 328, 338 (AIDS), 175,463 phate Acetone, 327-328 Acridine dyes, 440 , 364F Acetone powder, 51 Action potential, 164 Adenylylation, 349 Acetyl-ACP, 332-333 Activated complex, 87-89 Adipose tissue (depot fat), 308, 318 Acetylcholine, 164-165 Activation energy: see Energy of activation A-DNA, 182-183 Acety !cholinesterase, 89T, 96, 164-165 Activators, 94-95,471 ADP: see Adenosine 5 '-diphosphate Acetyl CoA, 276-278 Active acetaldehyde, 291 ADP-glucose, 267 in , 246, 250F, Active site, 84-86 Adrenal cortical , 156 267F,286F Active transport Adrenal gland, 209T formation from pyruvate, 278-279, 281F primary (pump), 162-163 Adrenaline: see Epinephrine free energy of hydrolysis, 278 secondary (cotransport), 163 Adrenocorticotropic hormone (ACTH), 209T in glyoxylate cycle, 288-289 Acyl carnitine: see Fatty acyl carnitine Adsorption chromatography, 511 in ketone bodies formation, 327-328 Acyl carrier protein (ACP), 330-334 Aerobes, 7 in , 321-339 Acyl CoA dehydrogenase, 323, 327F Affinity chromatography, 513-514 metabolic fates of, 327 Acyl CoA synthase: see Thiokinase Affinity labeling, 109 Acetyl CoA:ACP transacylase, 332-333 Acylglycerols, 143-145 AI CAR: see Aminoimidazole carboxamide Acetyl CoA carboxylase, 329-331, 335-336 Acylglycerol synthesis, 337 ribonucleotide Acetyl-coenzyme A: see Acetyl CoA Acyl transferase, 337F AICAR transformylase, 362F

Page numbers in boldface refer to structural formulas. F indicates a figure, T a table. Positional and configurational designations in chemical names are either omitted or disregarded in alphabetizing

549 550

AIDS (acquired immunodeficiency syn- Aminoimidazole succinylcarboxamide ribonu- Antiterrninator form of mRNA, 469F drome),463 cleotide (SAICAR}, 362 Antiviral compounds: see Cancer Am: see Aminoimidazole ribonucleotide ~-Aminoisobutyrate, 370 chemotherapy AIR carboxylase, 362F Aminopeptidase, 55 AP endonuclease, 441 Am synthetase, 362F Amino , 124 Aphidicolin, 429-430 Alanine, 28, 265-266 Amino terminus (N-terminus), 36, 55, 498 Apoenzyme, 94 pK~ values, 29T Aminotransferases,350-352 Apoprotein B-100, 149, 165-166 titration curve, 33F Ammonia, 347, 351-352 AP sites, 441 ~-Alanine, 41, 370F excretion,360,369 Apurinic site: see AP sites , 210T pK: value, 16T Apyrirnidinic site: see AP sites Albumin,67,210T,317 toxicity, 359 Arabinose, 118 Alcohol dehydrogenase, 206 Ammonia fixation, 346, 348-349 Arachidic acid, 143 Alcoholic : see Ethanol Ammonotelic organisms, 360 Arachidonic acid, 143 Aldaric acid, 122 AMP: see Adenosine 5 '-monophosphate Archaebacteria, 6-7 Aldehyde, 508T Amphibia, nitrogen excretion, 360 Arginase, 210T, 357-358 Aldimine, 350-351 Amphibolic pathway, 273 Arginine, 28 Alditol, 124 Amphipathic molecules, 20, 145 pK: values, 29T Aldolase, 242-244,251, 253F, 394F Ampholyte, 31 in urea cycle, 356-359 Aldonic acid, 122 Amphoteric compounds, 12, 14,31 Argininemia, 210T Aldose, 118 Amylase, 83, 115, 213 Argininosuccinase, 358F Aldosterone, 209T a-Amylase, 129-131 Argininosuccinate, 356-359, 358 Alkaline phosphatase, 195 ~-Amylase, 129-131 Argininosuccinate synthetase, 358F Alkaline reserve, 329 Amylopectin, 128-129 Arms, in tRNA, 413 Alkalosis, 14 a-Amylose, 128-129 Arnon, D. 1., 377 Allantoic acid, 369-370 Amytal,303F Arrhenius equation, 93-94 Allantoin, 369-370 Anabolic steroids, 156 Arrhenius plot, 94 Allolactose, 464 , 203-207 Arsenate, inhibition of glycolysis, 244 Allopurinol, 370-371 Anaerobes, 7 Ascorbate (), 122-123, 216T Allose, 118 Analbuminemia, 210T dietary requirement, 215T Allosteric effectors, 106-107 Analytical ultracentrifugation, 515 P/0 ratio for, 305 Allosteric enzymes, 106-108 Anaphylactic shock, 213 A site: see Arninoacyl site Alpha helix (a helix), 60-62 , 287-288 Asparagine, 28 Altrose, 118 Androgens, 156,209T pK~ values, 29T Amanita phalloides, 454 Anemia Asparagine synthase, 356 a-Amanitin, 454 hemolytic, 210T, 256 Aspartame (Nutrasweet), 42-43, 128T Amber codon, 416 sickle cell, 53, 210T Aspartate: see Aspartic acid Amethopterin: see Methotrexate macrocytic, 216T Aspartate transcarbamoylase (ATCase), 366 Amino acid activation, 48~85 pernicious, 216T Aspartic acid, 28, 352F Amino acid composition, 51-52 Anhydrides, 228 in malate-aspartate shuttle, 310-312 Amino acid oxidase, 352 starch: see Glycogen pK: values, 29T Amino acid residue, 36 Annealing, 192 in purine and pyrimidine synthesis, 362F, Amino acids, 27-45, 28 Anomeric carbon, 120 366F abbreviations, 29T Antenna , 383 in urea cycle, 356-359 acid-base properties, 30T, 31-35 Anthranilate synthase, 468F Aspirin (acetylsalicylic acid), 150 classification, 29, 31 Antibiotics, 41, 174,307-308 Astbury, W., 48 essential and nonessential, 31, 349 effects on protein synthesis, 494-496 ATCase: see Aspartate transcarbamoylase functional groups, 30 ionophorous, 160-161 Atherosclerosis, 165-166 glucogenic and ketogenic, 353 resistance to, 445, 494 Atkinson, D., 308 helix breaking, 60 Antibodies, 77-78 Atmosphere, primordial, 4 metabolism, 345-360 Antibody diversity, 470 ATP (adenosine 5' -triphosphate), 175 pK~ values, 29T Anticancer compounds: see Cancer in , 392-397 uncoded,421 chemotherapy in carbohydrate metabolism, 241-246,250, Amino acid sequence, 53-56 Anticoding (template) strand, 408F, 410 264-265 Aminoacyl adenylate (AA-AMP), 480, Anticodon, 410 in control of metabolism, 267F, 279,283, 483-484 Anti conformation, 173-174 286-287,367 Aminoacyl site (A site), 478-479 Antifolates, 369 in energy charge, 307-308 Aminoacyl-tRNA, 480, 483-484 Antigen , 78, 137 as energy-rich compound, 228-230 Aminoacyl-tRNA synthetases, 483-485 Antimetabolites, 102 free energy of hydrolysis, 224T p-Aminobenzoic acid (PABA), 102 Antimycin A, 303F in lipid metabolism, 320, 322,329-331, Aminoimidazole carboxamide ribonucleotide , 42, 154 341 (AICAR), 362 Antiparallel strands, 182 in membrane transport, 162-163, 329 Aminoimidazole ribonucleotide (Am), 362 Antiport, 163 in nitrogen metabolism, 347-348, 356-359 INDEX 551

ATP (adenosine 5'-triphosphate) (cont.) Bile, 213 Butyryl-ACP, 333-334

in nucleic acid metabolism, 362-363, 433, Bile acids, 155-156 B vitamins: see Vitamin B/B/B61B 12 435,455 Bile pigments, 355 Bypass, 264 in oxidative phosphorylation, 304-307 Bile salts, 155-156 in photophosphorylation, 386-390 Binding assay: see binding assay CAAT box, 455 in photorespiration, 397-399 Binding sites, 70--72, 78 Cacodylate buffer, 17 in , 483-485, 493-494, Biochemical equilibrium constant (K~; 0), 223, CAIR: see Carboxyaminoimidazole ribonu• 498 294 cleotide yield in aerobic metabolism, 309-312 Biochemistry, central themes of, xxv-xxvii Cairns, J., 428,431 ATPase: see Na+ -K+ ATPase Biocytin (biotinyllysine), 287, 330--331 Calcium, 155, 258 ATP cycle, 227 Biodegradable detergents, 146 dietary requirement, 21ST ATP synthase, 208T, 302F, 307, 386--387 Bioenergetics, 221-235 Calmodulin, 258 Attenuation, 469-470 Biological buffers: see Buffers Caloric value of foods, 50, 117, 141 Autocatalytic enzymes, 104 Biological evolution: see Evolution Calorie (cal), 18T Autocrine hormones: see Hormones Biological oxidation, 226 Calvin, M., 392 Autoradiography, 195, 518 Biomembranes, 9-10, 156--166 Calvin cycle, 392-397 Autotrophs, 7 asymmetry of, 159 carbon skeleton transformations in, 395F Auxotroph, 212 composition, 156-157 controlo~396-397 Avery, 0. T., 185 fluid mosaic model of, 157-159 stoichiometry, 396 Avery, McLeod, and McCarty experiment, transport processes in, 159-163 cAMP: see Cyclic AMP 185-186 Biotin, 21ST, 216T, 264,287, 326, 330--331 Cancer chemotherapy, 368-369 Axial bonds, 122 Biotin carboxylase, 330--331 CAP: see Catabolite activator protein Azaserine, 368-369 Biotin carboxyl carrier protein (BCCP), Cap-binding protein, 489 Azide, 303F 329-331 Capping enzyme, 459 3' -Azido-3' -deoxythymidine (AZT), 175, Biotinyllysine: see Biocytin Capping of mRNA: see Methylated cap 463 Birds, nitrogen excretion, 360 Capsid: see Protein coat 1,3-Bisphosphoglycerate, 229, 243-244, Capsule, bacterial, 135 Bacillus brevis, 40T 393-394 Carbamate, 348 Bacteriochlorophylls, 378T, 382 2,3-Bisphosphoglycerate (BPG), 75-76 Carbamino compounds, 75 Bacteriophage: see Phage Bisphosphoglycerate mutase, 244 Carbamoyl aspartate, 366 Bakers' yeast, 125T Biuret reaction, 51 Carbamoyl phosphate, 348 Balance study, 211 Blackman, F., 377 in ammonia fixation, 348-349 Baltimore, D., 462 Blobel, G., 496 in pyrimidine synthesis, 365-366 Basal factor, 471 Bloch, K., 339 in urea cycle, 356--359 Basal metabolic rate (BMR), 21ST Block: see Metabolic block Carbamoyl phosphate synthase I and II, 348, Base composition, 179, 191 Blood 357,365-366 Base pairing, 181 buffer systems in, 50 Carbohydrates, 117-140 Bases: see Purines, Pyrimidines glucose levels in, 238-239 chemical reactions of, 125-126 Base sequence determination, 194-197 Blood brain barrier, 327 digestion of, 213-214 Bassham, J., 392 Blood clotting, 58, 104, 154-155 energy value of, 117 Bathorhodopsin, 153F Blood group substances, 137 fermentability, 125T BCCP: see Biotin carboxyl carrier protein Blood sugar, 238-239 in , 133-134 B-DNA, 180--182 Boat conformation, 122-123 metabolism of, 237-271, 309-312 Beer's law, 511 Bohr effect, 74-75 transport and storage, 238-239 Benedict's reagent, 125 Bond energy, 18T See also Monosaccharides Benson, A., 392 Boundary, in sedimentation, 515-516 Benzoic acid, 320 BPG: see 2,3-bisphosphoglycerate from alcoholic fermentation, 206 Berg, P., 483 Braconnot, H., 27 effect on oxygen saturation curves, 74-75 Beriberi, 216T Brain, metabolism in, 263, 327 in photorespiration, 397-399 Berthelot, P., 83 Branching enzyme, 210T, 261 in photosynthesis, 392-397 Berzelius, J. J., 47 Branch migration, 444F in purine and pyrimidine synthesis, 362F, Beta bends: see Reverse turns Br!ilnsted acids and bases, 12, 14-18, 32-33 366F ([3 Oxidation), 321-327 pK: values, 16T Carbon dioxide fixation comparison with fatty acid synthesis, Brown, M.S., 165 in Calvin cycle, 392-397 335 Brown fat (brown adipose tissue), 308 in c3 and c4 plants, 399-401 energetics, 325-326 Buchner, E., 239 Carbonic acid, pK~ values, 16T regulation, 323 Buffers, 16-18 , 65F Beta pleated sheet ([3 sheet), 62-63 calculations, 504-505 kinetic parameters, 89T, lOOT , 329, 348, 356--359 pK: values, 17T Carbon monoxide, 30 I pK~ value, 16T Bundle sheath cells, 399-400 Carbonylcyanide-p-trifluoromethoxyphenyl• Bifunctional enzymes, 257 Burk, D., 99 hydrazone, 308 Big Bang theory, 3-4 a,[3-trans-Butenoyl-ACP, 333-334 Carbonyl phosphate, 348 552

Carboxyaminoimidazole ribonucleotide Chargaff's rules, 180 Citric acid cycle (cont.) (CAIR),362 Charge, estimation of net, 34-35 energetics, 281T, 285-286 Carboxybiocytin, 287 Charging of tRNA, 484 major features, 284-285 -y-Carboxyglutamate, 154-lSS Chase, M., 186 Citrulline, 356-359, 3S8 Carboxyl terminus (C-terminus), 36, 55 Chelonia, nitrogen excretion, 360 Clathrin, 165-166 Carboxypeptidase, 55 Chemical cleavage method: see Maxam- Cloning, 520--521 Carcinogens, 440 Gilbert method CMP: see Cytidine 5 '-monophosphate Camitine, 323 Chemical coupling hypothesis, 305 CoA: see Coenzyme A Carnitine acyl transferase, 324F Chemical evolution: see Evolution Coacervate droplets, 5 Carnitine carrier system, 322-324 Chemiosmotic coupling hypothesis, 305-307, Coactivator, 471 Carnosine, 40 386-387 Coated pits, 165-166 ~-, 151 Chemotherapy: see Cancer chemotherapy Cobalamin, 216T Carotenoids, 151,382-383 Chemotrophs, 8 Cobamide coenzymes, 216T Carotenosis, 152 Chiral center, 507 Coding (sense) strand, 408F, 410 Carriers: see Carnitine carrier system, Elec• Chi (X) structure, 444F Codons,410,415-416,419F tron carriers, Mobile carriers, Shuttle Chitin, 131 of mitochondria, 420T systems, Tricarboxylate transport system Chloramphenicol (chloromycetin), 49S Coenzyme A (CoA, CoA-SH), 216T, 276-278 Cascade mechanism, 104: see also Enzyme Chlorophylls, 378T, 382-384 in beta oxidation, 321-327 cascade antenna, 383 in fatty acid synthesis, 329-336 Casein, 67 special pair, 391 in multienzyme complexes, 279, 283 Catabolism, 203-207 , 5-6, 381-382 Coenzyme Q (CoQ, Q), 295--296, 325 Catabolite activator protein (CAP), 467-468 membrane composition, 156T Coenzymes, 94-95, 216T Catabolite repression, 468 in photorespiration, 397-398 Cofactor, 94-95 Catalase,309,352,398F Cholecalciferol (vitamin 0 3), 153 Cognate tRNA, 484 activation energy, 88T Cholesterol, ISS Cohesive ends, 193 kinetic parameters, 89T, I OOT absorption of dietary, 213 Cold-stable enzymes, 93 Catalysts, 84 in atherosclerosis, 165-166 Colicins, 520

Catalytic rate constant (k081), lOOT biosynthesis, 338-341 Collagen, 77 , 112 esters, 155 Committed step, 207 C4 cycle: see Hatch-Slack pathway in lipoproteins, 149T Common intermediate principle, 231-232 COP-choline, 337 Cholic acid, 1S6 Compactin, 338, 340 COP-ethanolamine, 337 Choline,l46, 165,318 Compartrnentation, 207-208 Cech, T. R., 460 Choline acety!transferase, 164-165 Competitive inhibition, 100--102 Cell coat, 137 Chondroitin sulfate, 132 Complementary base pairing, 181 Cell-free amino acid incorporating systems, Chromatin, 189 Complexes I-IV: see Respiratory complexes 417-418 Chromatography, 511-514 Complex lipids, 141, 146-150 Cell-free extract, 50 Chromatophore, 381 Complex polysaccharides: see Heteropolysac- Cell (plasma) membrane: see Biomembranes Chromatosome, 189 charides Cellobiose, 131 Chromium, dietary requirement, 215T Composite transposon: see Transposon Cells Chromoplasts, 189 Concanavalin A, helical content, 65T classifications, 7 Chromosomes, 187 Concerted model, 107-108 composition, 7T ONApackaging in, 187-189 Cone cells, 152 number of proteins in, 470 homologous, 444 Configuration, 509 prokaryotic and eukaryotic, 9-10 Chyle, 214 Conformation, 509 structure, 8-10 Chylomicrons, 149T Conformational coupling hypothesis, 305 , 226 Chymotrypsin Conformer, 509 Cellulase, 131 catalytic mechanism, 109-112 Coniferyl alcohol, 134 Cellulose, 130--131, 267 helical content, 65T Conjugate acid-base pairs, 14 Cell wall, 134-137 kinetic parameters, 89T, 1OOT Conjugated proteins, 49 Central dogma of molecular biology, 409, 462 specificity, 52T Conjugate redox pair, 523 Centrifugation,513,515-516 Citrate,86,281-282 Conjugation, 443-444 Cephalin, 147 in regulation of metabolism, 250F, 335 Connexin, 161-162 Ceramide, 147 in tricarboxylate transport system, 329 Consensussequence,455 Cerebrosides, 147-148 Citrate , 329 Constitutive enzymes, 464 CF0-CF1 ATPase: see ATP synthase , 208T, 281-282 Contact inhibition, 137 Chain terminator method: see Sanger-Coulson Citric acid: see Citrate Contour length, 178T method Citric acid cycle, 273-291 Control elements, 465 Chair conformation, 122-123 in amino acid metabolism, 353F Convergent pathways, 204 Changeux, J.P., 106 amphibolic nature of, 273-275, 353F Converter enzymes, 258 Channels, ligand and voltage gated, 161 anaplerotic reactions for, 287-288 Cooperative interactions, 71-74, 106-108, Chaperones, 496 controloL286-287 191-192 Chargaff, E., 180 discovery of, 273-274 Coordinate induction, 465 INDEX 553

Coordinate repression, 465 Cytochrome b6 , 386 Dextrose, 120 Copper Cytochrome bf complex, 386 , 210T, 238-239 in cytochrome oxidase, 299-300 Cytochrome c and fatty liver, 318-319 dietary requirement, 21ST absorption spectra, 298F and ketone bodies, 328 CoQ: see Coenzyme Q helical content, 65T Diabetes insipidus, 219 Cordycepin: see 3 '-deoxyadenosine unit evolutionary period, 58-60 I ,2-Diacylglycerol, 337 Co-repressor, 465-466 Cytochrome!, 386 1,2-Diacylglycerol3-phosphate, 337 Core protein, 133-134 Cytochrome/iron-sulfur complex, 386 Diacylglycerols, 143-144 Corey, R., 59 Cytochrome oxidase, 208T, 299-302 Dialysis, 49-50 Cori, C. and Cori G., 241, 247 Cytochrome reductase, 302T Diastase, 83 Cori cycle, 247-248 Cytochromes, 296-297 Diastereomers, 508 Corticotropin-releasing factor (CRF), 209T heme types, 297 2,6-Dichlorophenol indophenol, 315 Cortisol, 209T reduction potentials, 294T Dichlorophenyldimethylurea (DCMU), 403 Corynebacterium diphtheriae, 494 Cytoplasm, 8 2' ,3 '-Dideoxynucleoside triphosphates, 196 Cotranscriptional processing, 459 Cytosine, 172 Dideoxynucleotide method: see Sanger- Cotransport: see Active transport pK~ values, 173F Coulson method Coulomb's law, 20 Cytoskeleton, 10 Dielectric constant, II Coulson, A. R., 196 Cytosol, 9 Dienoyl CoA reductase, 326-327 Coupled reactions, 230-232 Dietary fiber, 215 Coupling hypotheses, 305-307 Dalton (D), 48 Dietary nutrients: see Nutrition, human Coupling of transcription and translation, Danielli, J., 157 Diets 469-470,482-483 Dansyl chloride reaction, 38-39 high fructose, 128, 252 Covalent catalysis, 90, 109-112 Dark reactions, 377, 392-397 low phenylalanine, 355 Covalently modifed enzymes I 06, 259F, 262F Davson, H., 157 megavitamin, 152, 217 Cozymase, 239 Deamination: see Oxidative deamination Diffusion, 158-162 c3 plants, 399-401 Debranching enzyme, 130, 257 Diffusion coefficient, 160 C 4 plants, 399-401 Deformylase, 496 Diffusion-controlled limit, I 00 Crabtree effect, 249-250 Degeneracy, 416,419--420 Digestion, 213-214 , 351 Dehydroascorbic acid: see Ascorbate Digestive fluids, 213 Crick, F., 180, 409, 413,421 7 -Dehydrocholesterol, 153 Diglycerides: see Diacylglycerols Cristae, 207-208 Dehydrogenases 7,8-Dihydrofolate, 367 Critical micelle concentration, 20 as flavoproteins, 276-277, 385 , 367, 369

Crossing over, 444 -linked, 275-276 Dihydrolipoyl dehydrogenase (E3), 279 C-terminus: see Carboxyl terminus Deisenhofer, J., 391 Dihydrolipoyl transacetylase (E2), 279 CTP: see Cytidine 5 '-triphosphate Deletion mutations, 439 , 366 Curie (Ci) of radioactivity, 518 Denaturation, 69-70 Dihydroorotate, 366 Cut, 185 thermal, of DNA, 190-192 Dihydroorotate dehydrogenase, 366 Cyanide, 30 I De novo synthesis, 361 Dihydropteridine reductase, 354-355 Cyanobacteria, 5-6, 378T, 381 Density gradient centrifugation, 515-516 Dihydropteroate synthetase, I 02 Cyanogen bromide reaction, 52 3' -Deoxyadenosine (cordycepin), 474 Dihydrosphingosine, 337 Cyclamate, 128 5 '-Deoxyadenosylcobalamin, 326 , 370 3' ,5' -Cyclic adenylic acid: see Cyclic AMP Deoxycholic acid, 156 5,6-, 172, 370F, 414 Cyclic AMP (cAMP), 176 Deoxyhemoglobin, 73F Dihydroxyacetone, 119 in catabolite repression, 467--468 Deox yribonucleases, 179-180 Dihydroxyacetone phosphate, 240, 243, 320, effect on carbohydrate metabolism, 250, Deoxyribonucleic acid: see DNA 337 258-259,262,266 Deoxyribonucleosides: see in Calvin cycle, 394F effect on HMG-CoA reductase, 339-340 : see Nucleotides in glycerol phosphate shuttle, 310-311 Cyclic electron flow, 389-391 , 125 I ,25-Dihydroxycholecalciferol, 153 Cyclic photophosphorylation, 390 Deoxysugars, 124 Diimine, 347 Cyclobutane ring, 440F Deoxythymidine: see Diisopropyl fluorophosphate, 95, 108-109 Cycloheximide, 495 Dephlogisticated air, 376 Dimethylallyl pyrophosphate, 338-339 Cyclooxygenase, 150 Depot fat: see Adipose tissue 2,4-Dinitrophenol, 308 Cysteine, 28 Depsipeptide, 160 Dinitrophenyl (DNP) amino acid, 36-37 pK: values, 29T Derived lipids, 141-142, 150-156 Dintzis, H., 480 Cystine, 509 Derived monosaccharides, 122-126 Dipeptide, 35 pK~ values, 33 Dermatan sulfate, 132 Diphtheria toxin, 494, 496 Cytidine, 174 Desaturases, 335-336 Dipolar ion, 31 Cytidine 5 '-monophosphate (CMP), 370 Detergents, 145-146 Dipole, II, 18,20-21 Cytidine 5 '-triphosphate (CTP), 367F Detoxification, 67, 321 Directionality ofreactions, 226, 295, 523-526 Cytidylic acid: see Cytidine 5' -monophosphate Dextrans, 129, 267-268 Disaccharides, 126-128 Cytochrome a: see Cytochrome oxidase Dextrins, 130 Disc gel electrophoresis, 517

Cytochrome a3 : see Cytochrome oxidase Dextrorotatory, 508 Discharged tRNA, 492 554 INDEX

Discontinuous DNA replication: see DNA DNA repair, 440-443 Electron transport system (cont.) replication DNA replication, 408, 425-451 sequence of carriers in, 298-301 Discontinuous (split) genes, 45~59 in eukaryotes, 431-432, 438 See also Oxidative phosphorylation Dismutation reaction, 309 fidelity of, 438-439 Electron transport systems Displacement loop (D-loop) replication, initiation of, 431, 438 of desaturases, 335-336 445-446 in mitochondria 445-446 in light activation systems, 396 Disproportionation reaction, 307 in prokaryotes, 431-438 of nitrogenase, 346 Dissociation constant, 14 rate of, 431 in photosynthesis, 384-385, 389, 391 Disulfide bonds in viruses, 446-447 of ribonucleotide reductase, 364-365 inter- and intrachain, 54 visualization of, 432F Electrophile, 507 in protein denaturation, 69 DNA-RNA hybrids, 193 Electrophoresis, 517-518 Disulfide exchange, 396 DNA synthesis: see DNA replication Electropositive, 11 Dithiothreitol, 42 DNA transcription: see Transcription Electrostatic interactions, 33, 90: see also Divergent pathways, 204 DNP-amino acid: see Dinitrophenyl amino Ionic interactions D-loop replication: see Displacement loop acid Elongases, 335 replication Dolichol, 268 Elongation factors 488T, 490-492 D,L-system, 509 Domains, 67 Embden, G., 241 DNA, 180-197 Donor site: see Peptidyl site Embden-Meyerhof pathway: see Glycolysis A, B, and Z forms, 180-184 Double helix, 180-184 Emerson, R., 377 amount per cell, 185T Double reciprocal plot, 99 Emerson enhancement effect, 377 coding and template strands, 408F, 410 Downhill reactions, 206-207,249 Emulsions, 145 denaturation, 189-192 Downhill reductions, 385 Enantiomers, 508 double helix, 180-184 Down regulation, 165 Endergonic reactions, 84, 206-207 eukaryotic, 178T, 187-188 Downstream, 454-455 End-group analysis, 36-40, 126 extrachromosomal, 189 Drosophila melanogaster (fruit fly), 178T, 438 Endocrine hormones: see Hormones linker (spacer), 189 dTMP: see Thymidine Endocytosis: see Receptor-mediated melting temperature (Tm), 191-192 Duclaux, E., 83 endocytosis packaging, 187-189 dUMP, conversion to dTMP, 367F Endoenzyme, 129 primary structure, 176-177 Dwarfism, 210T Endonucleases, 180, 193-194,441 prokaryotic, 178T, 187-188 Dynorphins, 42 Endopeptidases, 52 recombinant technology of, 518-521 Endoplasmic reticulum (ER), 9 relaxed, 184 Eadie-Scatchard plot, 115 Endorphins, 42, 209T repetitive,470-471 E. coli: see Endosome, 165-166 satellite, 470 Editing function: see Proofreading Endosymbiotic theory, 5-6 secondary structure, 180-184 Edman degradation, 39-40, 56 End-product inhibition, 105-106 sequencing of, 194-197 Edman reagent, 39 Energetically coupled reactions: see Coupled size, 178T Effectors: see Allosteric effectors reactions tertiary structure (superhelix), 184-185 EF-T (EF-Ts, EF-Tu): see Elongation factors Energetics: see Bioenergetics untranscribed, 470 Egg albumin, 67, 69 Energy, human requirement, 215T viral, 178T , 150 Energy charge, 307-308 dnaA protein, 431, 436T elF (eukaryotic initiation factors), 494 Energy of activation, 88, 93-94 dnaB protein: see Helicase Einstein, 379 Energy-rich bonds, 228 DNA-dependent DNA polymerase: see DNA Elastase, specificity, 52T Energy-rich compounds, 226-230 polymerase Electrochemical gradient, 306 Enhancer, 471 DNA-dependent RNA polymerase: see RNA Electrogenic pump, 163 Enkephalins, 40, 42, 209T polymerase Electromotive force, 294 , 245-246 DNA glycosylases, 441 Electron affinity, 293-294 Enol-keto tautomerism, 172-173 DNA gyrase, 184-185, 433, 436T Electron carriers Enoyl-ACP reductase, 333-334 DNA ligase, 434-435, 436T in photosynthesis, 384-385 trans-~ 2-Enoy1 CoA, 321, 323-324 DNA polymerase a (pol a), 429 reduction potentials of, 303-304 Enoyl CoA hydratase, 324 DNA polymerase I (pol 1), 89T, 427-428 in respiratory chain, 295-298 Enoyl CoA , 326-327 exonuclease activities of, 428 Electron density map, 64-65 3,2-Enoyl CoA isomerase, 327 Klenow fragment of, 428 Electronegativity, 11 Enteropeptidase, 103 processivity, 42~29 Electron holes, 385-387 Entropy, 18-20,88-90 DNA polymerase II (pol II), 42~29, 436T Electron transfer, 379-380 Enzyme cascade, 104 DNA polymerase ill (pol ill), 42~29, 436T Electron-transfer flavoprotein (ETF), 325 in fatty acid mobilization, 318-319 holoenzyme, 429T Electron transport system (ETS, respiratory in , 262 purification of, 90T chain), 293-305 in , 258-259 DNApolymerases, 408-409,426-430 energetics,303-305 Enzyme commission (EC), 86 eukaryotic,429-430 inhibitors of, 302-303 Enzymeinduction,464-468 prokaryotic,427-429 respiratory complexes of, 284, 301-302 Enzyme inhibition: see Inhibitors INDEX 555

Enzyme-inhibitor complex, 95 Estrogens, !56, 209T Fatty acids, 142-143 , 96-103 Estrone, 209T activation of, 321-322 Enzyme repression, 465-470 Ethanol beta oxidation, 321-327 Enzymes, 83-116 from alcoholic fermentation, 206 essential, 143, 335 activity, measures of, 89, 100 effect on gluconeogenesis, 266 mobilization, 318-319 allosteric, I 06-108 Ethanolamine, 146 naturally occurring, 143 classification, 86-87 Ethidium bromide, 440 peroxidation, 154 covalently modified, 106, 259F, 262F ETS: see Electron transport system serum albumin complexes, 317 effect of light on, 396-397 Eukaryotes unsaturated, 142-143, 158, 326-327 efficiency, 89-90 comparison with prokaryotes, 9-10 complex, 330, 332 induced fit of, 85 DNAof, 178T, 185T, 187-188 Fatty acid synthesis, 329-336 lock and key theory of, 84-85 DNA replication, 431--432, 438 committed step, 329 polyaffinity theory of, 85-86, 282 gene regulation, 4 70--4 72 comparison with beta oxidation, 335 properties, 91-96 organelles of, 9T regulation, 335-336 purification, 90T protein synthesis, 489 Fatty acyl adenylate (fatty acyl-AMP), 322 regulatory, 105-108 ribosomes of, 412T Fatty acyl carnitine, 323 restriction, 193-194 Euler, U., 150 Fatty acyl CoA, 321-323 specificity, 84-89 Evolution Fatty liver, 318-319 Enzyme-substrate complex, 84 biological, 5-7 Fd:NADP+ reductase (FNR), 385 Enzyme-substrate compound, 90 chemical, 3-5 FDNB: see 1-fluoro-2,4-dinitrobenzene Enzyme units, 89 of photosynthesis, 378 Feasibility of reactions, 232 Epimeric carbon, 120 Evolutionary tree, 6F, 58 Feedback inhibition, 105-106 Epinephrine (adrenaline), 209T, 355 Excision repair, 440--442 Fehling's reagent, 125 in fight or flight response, 258-259 Excited state, 379-380 Fe-protein, 346-347 in regulation of metabolism, 238, 259F, Exciton transfer: see Resonance energy Fermentation, 204 262F,336 transfer alcoholic, 206 Episomes, 188 Exergonic reactions, 87, 206-207 of carbohydrates, 125T Equal: see Aspartame Exhaustive methylation, 126 lactate, 206 Equatorial bonds, 122 Exocytosis, 164 Ferredoxin,346,385,396 Equilibrium constant, 12 Exoenzyme, 129 reduction potential, 294T biochemical (K~; 0 ), 223, 294 Exons, 416 Ferredoxin-thioredoxin reductase, 396 Ergocalciferol (vitamin D2), 153-154 Exon splicing, 458--459 Ferricyanide, 303F. Ergosterol, 153-154 Exonuclease, 180 Ferritin, 49T Erythroblastosis fetalis, 137 3'->5' Exonuclease, 428--430 Fe-S proteins: see Iron-sulfur proteins Erythrocytes 5'->3' Exonuclease, 428-430 Fetal hemoglobin, 76

blood group substances of, 137 Exopeptidase, 52, 55 F0 F1-ATPase: see ATP synthase DNA content, 185T Extinction coefficient, 511 FGAM: see Formylglycinamidine ribonu- glutathione requirements, 255-256 Extrachromosomal DNA, 189 cleotide membrane composition, 156T Extrinsic proteins, !58 FGAM synthetase, 362F Erythromycin, 495 FGAR: see Formylglycinamide ribonucleotide Erythrose, 118 Facilitated (mediated) transport, 160-162 Fiber: see Dietary fiber

Erythrose 4-phosphate, 255, 394F FAD/FADH2 (flavin adenine dinucleotide), Fibrinopeptides, 58, 60F Erythrulose, 119 276-277,352 Fibrous proteins, 50, 65 Escherichia coli in beta oxidation, 323 Fidelity cell composition, 7T in citric acid cycle, 283-284 in DNA replication, 438--439 DNA of, 178T, 185T in electron transport system, 301 in translation, 485, 490 DNA polymerase III purification, 90T in multienzyme complexes, 279, 283 Fight or Flight response, 258-259 excision repair in, 441 oxidation-reduction of, 277 Filmer, D., 106 generation time of, 211 reduction potential, 294T Fingerprint: see Peptide maps, Nucleotide his operon, 474 in ribonucleotide reduction, 364-365 maps lac operon, 467-468 FAICAR: see Formaminoimidazole carbox- First messenger, 208 protein synthesis rate, 4 77 amide ribonucleotide First order reaction, 96 ribosomes, 412T Familial hypercholesterolemia, 166 Fischer, E., 117 RNA content of, 411, 413 Faraday constant (F), 294 Fischer projections, 120, 146 transcription rate, 474 Farnesyl pyrophosphate, 339 Fish, nitrogen excretion, 360 trp operon, 468-470 Fasting, 266-267 Flagella, 9

Essential amino acids, 31, 349 Fats, 142-145,318-320 Flavin adenine dinucleotide: see FADIFADH2 Essential fatty acids, 143, 335 brown, 308 Flavin coenzymes, 216T, 276-277

Estimated safe and adequate daily dietary digestion of, 213-214 Flavin mononucleotide: see FMNIFMNH2 intake (ESADDI), 21ST energy value, 141 Flavoproteins, 276-277 Estradiol, 156 Fat-soluble vitamins, 151-155 Flexible active site: see Induced fit model 556 INDEX

Flickering cluster model, 12 Fructose 1,6-bisphosphate, 240, 394F Genes (cont.) Flip-flop (transverse diffusion), 158-159 in gluconeogenesis, 264-265 structural and regulatory, 465 Fluid mosaic model, 157-159 in glycolysis, 242 transposable, 444-445 Fluorescence,379-380 Fructose 2,6-bisphosphate Gene therapy, 211 Fluoride, 245 in regulation of metabolism, 250F, 266 Genetic code, 415-421 dietary requirement, 215T Fructose !-phosphate, 251 characteristics, 419-421 Fluoroacetate, 282 Fructose 6-phosphate, 240,251-252, 268 deciphering of, 417-419 Fluoroacetyl CoA, 282 in gluconeogenesis, 265 dictionary, 419F Fluorocitrate, 282 in glycolysis, 242 evolution of, 420-421 5-Fluorodeoxyuridylate, 369 in photosynthesis, 394F of mitochondria, 420T 1-Fluoro-2,4-dinitrobenzene (FDNB), 36-37 Fruit fly (Drosophila), 178T, 438 Genetic diseases, 209-211 5-Fluorouracil, 368-369 , 208T, 284 in carbohydrate metabolism, 210T, 255, 263 Flush cuts, 193 kinetic parameters, I OOT in lipid metabolism, 338 fMet-tRNArr•• (fMet-tRNNM••): see Fumarate, 280, 354 in nucleic acid metabolism, 441-442 N-formylmethionyl-tRNA in citric acid cycle, 284 in protein metabolism, 210T, 354-355 FMN/FMNH2, 276-277, 352 in urea cycle, 357-358 Genetic engineering: see Recombinant DNA in electron transport system, 299-301 : see Fumarate technology oxidation-reduction of, 277 Fumarylacetoacetate, 354 Genetic recombination, 443-444 reduction potential, 304T Functional groups, 507-508 Genome, 9 Folate coenzymes, 102, 216T, 361-363,486 Funk, C., 216 Geranyl pyrophosphate, 339 Folic acid, 102, 216T, 363 Furan, 122 Gibb's free energy: see Free energy change dietary requirement, 215T Furanoside, 122 Gilbert, W., 194, 467 Follicle-stimulating hormone (FSH), 209T Fusidic acid, 495T Globin, 58, 60F, 71 Foods, caloric value, 50, 117, 141 Futile cycle, 207 Globular proteins, 50, 65-66 Formaminoimidazole carboxamide ribonu- in nitrogen fixation, 347 Glucagon, 209T cleotide (FAICAR), 362 effect on blood sugar, 238 Formate Galactokinase, 210T, 252 in regulation of metabolism, 259F, 262F, pK~ value, 16T Galactose, 118 336,340 in purine synthesis, 362F catabolism, 252-253 Glucans, 128 Formylglycinamide ribonucleotide (FGAR), · fermentability, 125T Glucaric acid, 123 362 sweetness, 128T Glucocerebrosidase, 210T Formylglycinamidine ribonucleotide (FGAM), Galactosemia, 210T, 253 Glucocorticoids, 156, 209T 362 Galactose !-phosphate, 252 Glucogenic amino acids, 353 N-Formylmethionine, 486 Galactose !-phosphate uridyl transferase, Glucokinase, 242, 259 N-Formylmethionyl-tRNA (fMet-tRN~Met, 253 Gluconeogenesis, 263-267 fMet-tRNAfMet), 486 13-Galactosidase, 464, 467-468 effect of alcohol on, 266 N10-Formyltetrahydrofolate, 362-363, 486 turnover number, 89T regulation of, 266-267 Fractional precipitation, 69 13-Galactoside permease, 465 Gluconic acid, 123 Frameshift mutations, 439 Galactosyl transferase, 268 Glucosamine, biosynthesis, 268 Franklin, R., 180 Galacturonic acid, 134 Glucose,118,237-238,263 Free energy change, 221-226 Gamov, G., 415-416 ATP yields from, 309-312 actual conditions, 223-225 Gangliosides, 147-148 blood levels of, 238-239 of ATP hydrolysis, 228-230 Gap junction, 161-162 in catabolite repression, 468 of biochemical reactions, 224T GAR: see Glycinamide ribonucleotide fermentability, 125T effects of variables on, 225-226 GAR synthetase, 362F in glycolysis, 239-241 from entropy and enthalpy, 88, 225 GAR transformylase, 362F o:/13-isomers, 120-121 from equilibrium constants, 222-223 Gas constant (R), 222 membrane transport of, 241 and reaction rate, 222 Gastric juice, 213 mutarotation, 120-122 from reduction potentials, 295 Gastrin, 40 sweetness, l28T standard conditions, 222-224 Gated channels, 161 Glucose-alanine cycle, 265-266 Free energy of ionization, 222-224, 229-230 Gaucher's disease, 210T Glucose 6-phosphatase, 265 Freeze drying, 51 GOP: see Guanosine 5'-diphosphate Glucose !-phosphate, 252, 257, 260 Freeze fracture technique, 157 Geiger-Mueller counting, 518 Glucose 6-phosphate Frictional coefficient, 517 Gel-filtration chromatography, 513-514 in anabolism, 259-265 Frozen accident school, 420 Gene amplification, 472 in catabolism, 241-242,252-257 Fructokinase, 251 Genes, 465 Glucose 6-phosphate dehydrogenase, 210T, Fructose, 119, 267 cancer causing (oncogenes), 464 253-256,397 catabolism, 251-252 cloning, in recombinant DNA technology, Glucose tolerance curve, 238-239 fermentability, 125T 518-521 o:-1,4-Glucosidase, 210T sweetness, 128T discontinuous (split), 458-459 Glucuronic acid, 123 Fructose 1,6-bisphosphatase, 253F, 265-266, regulation in eukaryotes, 470-472 Glutamate: see Glutamic acid 394F regulation in prokaryotes, 464-470 , 348, 352 INDEX 557

Glutamic acid, 28, 352 Glycolysis (cont.) Half-life (cont.) in ammonia fixation, 348 entry of carbohydrates into, 250-253 of proteins, 497--498 biosynthesis, 348 light inactivation in, 397 radioactive, 518 pK: values, 29T regulation of, 242, 249-250, 266--267 Half-reactions, 523 titration curve, 33F Glycoproteins, 133-134, 268 Halobacterium, 156T Glutamine, 28, 268, 362F, 366F Glycosaminoglycans, 132-133 Halophiles, 7 biosynthesis, 230-231, 348 Glycosidases, 131 Harden, A., 239 pK~ values, 29T Glycosidic bonds, 122, 130, 172 Hard soaps, 144 Glutamine antagonists, 369 Glycosylase: see DNA glycosylase Hatch, M., 399 Glutamine-PRPP amidotransferase, 361-362, , 133 Hatch-Slack pathway, 399--401 369 Glyoxylate cycle, 288-289 Haworth, N., 126 Glutamine synthase, 231, 348-349, 356 , 288, 369, 398F Haworth projections, 122 -y-Glutamyl phosphate, 231 Glyoxysomes, 10 HDL (high-density lipoproteins), 149T, 166, Glutaredoxin, 365 GMP: see Guanosine 5'-monophosphate 210T Glutathione Goldstein, J. L., 165 Head-to-tail polymerization, 151, 340 functions in red blood cells, 255-256 Golgi apparatus, 9-10, 268,497 Heart attack, 351 oxidation-reduction of, 40--42, 309, 365 Gonadotropin-releasing factor (GnRF), 209T Heat of vaporization, 12 Glutathione peroxidase, 309 Gonads, 209T Helical content, proteins, 65T Glutathione reductase, 42, 255, 365 Good's buffers, 16 Helicase (dnaB protein), 433, 436T , 128 Gorter, E., 157 a-Helix: see Alpha helix Glyceraldehyde, 118, 251 Goulian, M., 186 Helix-breaking amino acids, 60 stereoisomers (D,L; R,S), 509-510 Gout, 370-371 Helix pitch, 61F, 183T Glyceraldehyde 3-phosphate G proteins, 258, 493 Helix rise, 183T in Calvin cycle, 393-394 Gramicidin A, 161, 308 Helix-tum-helix motif, 471 in glycolysis, 240, 242-244 Gramicidin S, 40--42 Helper T cells, 463 Glyceraldehyde 3-phosphate dehydrogenase, Gram-negative organisms, 134-135 Heme, 297, 356F 68F,243-245,394F Gram-positive organisms, 134-135, 156T Heme-controlled inhibitor, 494 Glycerate, 398 Grana, 381 Hemiacetal, 120-121 Glycerides: see Acylglycerols Gratuitous inducer, 465 Hemicellulose, 134 Glycerokinase, 320 Grendel, F, 157 Hemiketal, 120-121 Glycerol, 86, 143-144, 337 Griffith, R., 185 Hemoglobin, 48T, 210T degradation, 319-320 Ground state, 379-380 fetal, 76 Glycerol 3-phosphate Group translocation, 163 helical content, 65T in fat metabolism, 320, 337 Growth hormone (GH), 209T, 210T oxygen binding by, 70-76 in glycerol phosphate shuttle, 310-311 Growth hormone-releasing factor, 209T peptide maps of, 53F Glycerol phosphate dehydrogenase, 310-311 GTP: see Guanosine 5 '-triphosphate quaternary structure, 73F Glycerol phosphate shuttle, 310-311 GTPase (GTP ), 258 sickle cell, 53-54 Glycerophospholipids, 146--147, 337-338 Guanidine hydrochloride, 70 synthesis rate, 4 77 Glycinamide ribonucleotide (GAR), 362 Guanido group, 30 unit evolutionary period of, 60F Glycine, 28, 156F, 362F, 398F , 172 x-ray diffraction of, 64-65 in detoxification, 321 in G proteins, 258, 493 Hemoglobin A, 53 pK: values, 29T in methylated caps, 459--460 Hemoglobin S, 53 in reverse turns, 63 pK~ values, 173F Hemolytic anemia: see Anemia Glycocalyx, 137 , 368F Henderson-Hasselbalch equation, 14-15 Glycogen, 129 Guanosine, 368 calculations, 503-504 biosynthesis, 259-263 Guanosine 5 '-diphosphate (GDP), 258, 493 Henri, V., 92 degradation, 129-130, 256--259 Guanosine 5 '-monophosphate (GMP), 361, Henseleit, K., 274, 356 fermentability, 125T 364F, 368 Heparan sulfate, 133 Glycogenesis, 259-263, 266-267 Guanosine 5 '-triphosphate (GTP), 264, 336 Heparin, 132 Glycogenic amino acids, 353 from citric acid cycle, 283-284 Heptose, 119 Glycogenin, 260-261 in G proteins, 258, 493 Hereditary diseases: see Genetic diseases Glycogenolysis, 256--259, 266--267 in protein synthesis, 488--494 Hershey, A. D., 186 Glycogen phosphorylase, 106, 256-257 in transcription, 455, 459 Hershey-Chase experiment, 186--187 Glycogen storage diseases, 210T, 263 Guanylic acid: see Guanosine 5'-monophos• Heteropolysaccharides, 131-137 Glycogen synthase, 260-263 phate Heterotrophs, 7 Glycolate, 398 Gulose, 118 Heterotropic interactions, 106-107 Glycolipids, 147-149 Gyrase: see DNA gyrase Hexokinase, 48T, 241-242, 259 Glycolysis, 239-253, 266-267 Hexose, 119 aerobic/anaerobic conditions, 246--248 Hairpin (stem and loop), 456--457 High-density lipoproteins: see HDL committed step, 242 Hales, S., 376 High-energy bonds: see Energy-rich bonds end products, 246 Half-life High-energy compounds: see Energy-rich energetics, 241 T, 248-249 ofmRNA, 411,467 compounds 558 INDEX

High-fructose diets, 128, 252 Hydroperoxyl radical, 309 Inorganic phosphate (Pi), 125 High-performance liquid chromatography Hydrophilic and hydrophobic groups, 19-20 Inosine, 368, 414 (HPLC), 513 Hydrophobic interactions, 18-20 Inosine 5 '-monophosphate (IMP), 360-364, Hill, R., 377 in DNA, 181-182 362,368 Hill reaction, 377 in proteins, 65-66 Inositol: see myo-inositol Hippuric acid, 320 13-Hydroxyacyl-ACP , 333-334 Insertion mutations, 439 his operon, 474 L-3-Hydroxyacyl CoA, 324 Insertion sequence (IS), 445 flisturUne,213,355-356 L-3-Hydroxyacyl CoA dehydrogenase, 324 Insulin, 36, 48T, 65T, 209T, 210T Histidine, 28, 109 13-Hydroxybutyrate, 327-328 effect on carbohydrate metabolism, 238, pK: values, 29T 13-Hydroxybutyrate dehydrogenase, 328F 262 titration curve, 33F D-13-Hydroxybutyryl-ACP, 333-334 effect on enzymes, 95 Histones, 81, 188-189 25-Hydroxycholecalciferol, 153 effect on lipid metabolism, 328, 336, 340 biosynthesis, 438 N-2-Hydroxyethylpiperazine-N' -2-ethanesul- Insulin-dependent (independent) diabetes, HIV: see Human immunodeficiency virus fonic acid (HEPES), 17 238-239 HMG-CoA (3-hydroxy-3-methylglutaryl Hydroxyisovaleric acid, 161 Integral proteins, 158 CoA), 338-340 Hydroxyl radical, 42, 154, 308-309 Intercalating agents, 440 HMG-CoA lyase, 328F Hydroxylysine, 30 Interferons, 494 HMG-CoA reductase, 338-340 3-Hydroxy-3-methylglutaryl CoA: see HMG- Intergranallamellae, 381 HMG-CoA synthase, 328F, 338F CoA Intermediary metabolism, 203 Hogness box, 455 p-Hydroxyphenylpyruvate, 354 Intermediate-density lipoproteins: see IDL Holley, R., 194 Hydroxyproline, 30, 60 Internal conversion, 379-380 Holliday, R., 444 Hydroxypyruvate, 398 Internalization of receptors: see Receptor- Holliday intermediate, 444F 5-Hydroxytryptamine (serotonin), 355-356 mediated endocytosis Holoenzyme, 94 Hyperbolic binding curve, 70, 91-92 Interspersed repeats, 4 70 Homogenate: see Tissue preparations Hyperchromic effect, 190-192 Intestinal fluid, 213 Homogentisate, 354 Hypochromic effect, 190 Intrathylakoid space: see disks Homologous chromosomes, 444 Hypoglycemia, 266 Intrinsic proteins, 158 Homology: see Sequence homology Hypothalamus, 209T lntrons, 416 Homopolynucleotides, 417 , 172 Invertase, 128 Homopolypeptides, 417 Hypoxanthine-guanine phosphoribosyl trans• Inverted repeats, 193, 445 Homopolysaccharides, 128-131 ferase (HGPRT), 373 Invert sugar, 128 Homoserine, 52 in vitro/in vivo, 211 Homotropic interactions, 106-107 Ice structure, 12 Iodination, 142 Hoppe-Seyler, E., 171 IDL (intermediate-density lipoproteins), 149T Iodine Hormones, 40T, 208-210 Idose, 118 dietary requirement, 215T in carbohydrate metabolism, 238, 259, 262 IF: see Initiation factors starch complex, 129 classification, 208 lg: see Immunoglobulins Iodine number 142-143 effects on enzymes, 95 Imidazole group, 30 Iodoacetamide,95 in fatty acid mobilization, 319F Immunoglobulins (lg), 77-78 Ion-exchange chromatography, 33-34, human, 209T IMP: see Inosine 5' -monophosphate 512-513 interactions with cells, 257-258 IMP cyclohydrolase, 362F Ionic interactions, 18T, 20-21 HPLC: see High-performance liquid ring, 30 in proteins, 62 chromatography Induced fit model, 85, 242 See also Electrostatic interactions Huber, R., 391 Inducer, 464-466 Ionic strength Human immunodeficiency virus (HIV), 463 Inducible enzymes, 464 calculation, 503 Hyaluronic acid, 132 Induction effect, 21 effect on DNA, 190 Hyaluronidase, 132 lngen-Housz, J., 376 effect on pK:. 15-16 Hybridization, 192-193 Ingram, V., 53 effect on proteins, 67-70 Hydrazine, 55-56, 347 Inhibitor constant (Ki), 101 Ionization, Hydride ion (H-) transfer, 243-244, 276 Inhibitors of biomolecules, 14, 30T, 173F Hydrochloric acid, in stomach, 213 competitive, 100-102 free energy of, 222-224, 229-230 Hydrogenation, 142 irreversible, 96 Ionophores, 160-161 Hydrogen bonds, 11, 18 noncompetitive, 101-103 Ion product of water (~). 13 in DNA, 181 reversible, 95 Iron, dietary requirement, 215T in proteins, 59-63 uncompetitive, lOlF, 103 Iron-porphyrin complex: see Heme in RNA, 412-414 See also Feedback inhibition Iron-sulfur proteins, 296, 298 in water, 11-12 Initial velocity, 91-92, 97 IS: see Insertion sequence Hydrogen electrode, 294 Initiation codon, 486 Isoacceptor tRNAs, 413 Hydrogen peroxide, 42, 88T, 154, 308-309, Initiation complex in translation, 488-489 Isoalloxazine ring, 277 352 Initiation factors (IF), 487-489 Isocitrate, 280, 288 , 87T Initiator (primer), 409 , 282-283 Hydronium ion, 12 Initiator tRNA, 486, 489 Isocitrate lyase, 288 INDEX 559

Isoelectric focusing, 32 Kidneys, metabolism in, 23, 152-153, 251, Levorotatory, 508 Isoelectric point (pi), 32 352 Liebig, J., 83 albumin, 67 Kilobase (kb ), 178T Ligand, 70 amino acids, 32 Kilocalorie (kcal), 18T Ligand-gated channel, 161 casein, 67 Kilojoule (kJ), 18T , 87T: see also DNA ligase estimation of, 35, 44 Kinase, 241 Light lysozyme, 80 Klenow fragment, 428 absorption of, 379-380, 511 myoglobin, 80 Km: see Michaelis constant photon energies, 379 urease, 80 Knoop, F., 273, 320 Light activation/inactivation, 396-397 Isoenzymes: see Isozymes Knoop's hypothesis, 320--321 Light reactions, 377, 384-391 Isoleucine, 28 Kornberg, A., 186,427 Lignin, 134 pK: values, 29T Kornberg, H.R., 274, 288 Limit dextrin, 130 , 87T Kornberg enzyme: see DNA polymerase I Lineweaver, H., 99 Isomorphous replacement, 65 Koshland, D. E., 106 Lineweaver-Burk transformations, 99, 101, Isopentenyl pyrophosphate, 151, 338-339 Krebs,H.,273-274,288,356 103 Isopentenyl pyrophosphate isomerase, 338F Krebs cycle: see Citric acid cycle Linked reactions: see Coupled reactions

Isoprene, 151,295 K5 : see Substrate constant Linker (spacer) DNA, 189 Isoprenoids, 151 Kiihne, W., 83 Link protein, 133-134 Isopropylthiogalactoside (IPTG), 464--465 ~: see Ion product of water Linoleic acid, 143 Isotopes, radioactive, 211-212,518 Kwashiorkor, 345 Linolenic acid, 143 Isozymes (isoenzymes), 104 Lipases, 317-318 Label, radioactive, 211-212,518 Lipid A, 134 Jacob, F., 411,465 lac operon, 467-468 Lipid bilayer, 157-158 Jagendorf, A., 387 lac repressor, 467 Lipids, 141-169 Joule (J), 18T a-Lactalbumin, 268 classification, 141-142, 145 Lactase, 127, 210T digestion of, 213-214 K~: see Acid dissociation constant Lactate, 247-248 energy value of, 141 Kaempfer, R. 0. R., 479 fermentation, 206 metabolism of, 317-344 Kamen, M., 377 pK: value, 16T transport and storage, 317-319 Kaplan, N., 274 Lactate dehydrogenase, 206,246, 351,497 Lipid storage diseases, 21 OT, 338 kb: see Kilobase isozymes, 104, 248 Lipmann, F., 274 K~io: see Biochemical equilibrium constant kinetic parameters, 89T, 248 Lipoarnide: see Lipoyllysine kcat: see Catalytic rate constant Lactic acid: see Lactate Lipoic acid, 216T, 278, 283 kca/Km: see Specificity constant Lactone, 52, 254F Lipopolysaccharides, 134 Kendrew, J., 64 Lactose, 127 Lipoproteins, 149T Kennedy, E., 293 biosynthesis, 268 Liposomes, 5, 157-158 Keratan sulfate, 132 fermentability, 125T Lipotropic agents, 318 Keratin, 76-77 sweetness, 128T Lipoyllysine (lipoamide), 216T, 278 Ketal, 120--121 Lactose intolerance, 127, 210T Liquid scintillation counting, 518 Ketimine,350--351 Lactose synthase, 268 Lithium borohydride, 55-56 Keto acid, 350--352 Lagging strand, 434 Liver 13-Ketoacyl-ACP reductase, 333-334 : see Phage damage to, 318-319,351 13-Ketoacyl-ACP synthase, 332-333 Lanosterol, 340--341 membrane composition, 156T 13-Ketoacyl CoA, 321, 324-325 Lariat, 459 metabolism in, 247F, 265F, 266-267, 352 Ketogenic amino acids, 353 Lateral diffusion, 158-159 in formation, 153 a-Ketoglutarate Lauric acid, 143 Living matter, xxvii in citric acid cycle, 280, 282-283 Lavoisier, A., xxv Lobry de Bruyn-Alberta van Eckenstein trans- in nitrogen metabolism, 348, 352 LDL (low-density lipoproteins), 149T, formation, 120 a-Ketoglutarate dehydrogenase complex, 165-166,210T Lock and Key theory, 84-85 283 LDL receptors, 165-166 London dispersion forces, 21 Ketone, 508T Leadersequence,469 Loops, in tRNA, 413 Ketone bodies, 327-329, 328 Leading strand, 434 Lovastatin, 338, 340 Ketonemia, 328 Leaflet: see Monolayer Low-density lipoproteins: see LDL Ketonuria, 328 Lecithin, 147 Lowry reaction, 51 Ketose, 119 Leder, P., 418 Lumen: see Thylakoid disks Ketosis, 210T, 328-329 Left-handed helix, 183-184 Lungfish, 178T,360 3-Ketosphinganine, 337 Lehninger, A., 293 Luteinizing hormone (LH), 209T 3-Ketosphinganine reductase, 337F Lesch-Nyhan syndrome, 373-374 , 87T 3-Ketosphinganine synthase, 337F Leucine, 28 Lynen, F., 274 13-Ketothiolase: see Thiolase pK: values, 29T Lyophilization, 51 Khorana, G., 418 Leu-enkephalin, 40 Lysine, 28 Ki: see Inhibitor constant Leukotrienes, 151 pK~ values, 29T 560 INDEX

Lysosomes,9-IO, 103 Messenger RNA, 410--411, 481--483 Microminerals, 214 Lysozyme, 135 amount per cell, 411 Micronutrients, 214 helical content, 65T capping of, 459-460 Microspheres, 5 isoelectric point, 80 half-life, 411,467 Microtrabecular network, 10 Lyxose, 118 mono- and polycistronic, 467, 471 Microtubules, 10 polyadenylation of, 460--461 Miescher, F., 171 MacLeod, C. M., 185 processing of, 458--460 Milk sugar: see Lactose Macrominerals, 214 size, 179T Miller, S. L., 27 Macromolecules, 48 synthetic, 417--419 Mineralocorticoids, !56, 209T Macronutrients, 214 Metabolic block, 211 Minimum molecular weight, 38, 48--49 Magnesium Metabolic pathways, 206-207, 327 Minor groove, 182 ATP complex with, 241 Metabolism Misincorporation dietary requirement, 21ST of amino acids and nucleotides, 345-374 of amino acids, 485, 490 Major groove, 182 of carbohydrates, 231-271 of nucleotides, 438--439 L-Malate, 280, 284, 288, 400F compartrnentation of, 207-208 Mismatch repair, 442 in malate-aspartate shuttle, 310--312 energy relationships in, 226-227 Missense mutations, 421 in tricarboxylate transport system, 330F intermediary, 203 Mitchell, P., 305 Malate-aspartate shuttle, 310-312 introduction to, 203-219 Mitochondria, 207-208 , 284, 311F, 330F oflipids, 317-344 DNA replication in, 445--446 Malate synthase, 288 pathways of: see Metabolic pathways evolutionary origins of, 5--6 Maleylacetoacetate, 354 regulation of, 206-211, 262-263, 266-267, genetic code of, 420T , pK~ 16T 307-308 in glyoxylate cycle, 288-289 Malic enzyme, 291, 330F study of, 211-213 membrane composition of, 156T Malonate, 284 Met-enkephalin, 40 in photorespiration, 397-398 Malonyl-ACP, 332-333 Methanogens, 7 transport systems of, 310--312, 322-324, Malonyl CoA, 323, 329-333, 331 Methionine, 28, 318 329-330 Malonyl CoA:ACP transacylase, 332-333 pK~ values, 29T in urea cycle, 358F Maltose, 126-127 in translation, 486--487 Mitomycin C, 440 fermentability, 125T Methotrexate, 368-369 Mixed-function oxygenase, 354-355 sweetness, 128T NI-Methyladenine, 172 Mobile carriers, 160, 295, 387F , nitrogen excretion, 360 Methylases: see Modification methylases Mobile genetic elements, 444-445 Manganese, dietary requirement, 21ST Methylated cap, 459-460 Mobilization: see Fatty acids, mobilization Manganese-containing Methylation: see Exhaustive methylation Modification methylases, 193-194 (MnC), 386 5-Methylcytosine, 172 Modified bases, 172, 414--415 Mannose, 118,252 Methylene blue, 303F MoPe-protein, 346-347 Mannose 6-phosphate, 252 N5,N10-Methylenetetrabydrofolate, 363, 367, Mole percent (mol%), 51 Marker enzymes, 208 369 Molecular biology, 407, 409 Martins, C., 273 7-Methylguanosine, 460 central dogma of, 409 Matrix,207 Methylmalonic acidemia, 210T introduction to, 407--424 Matthaei, H., 417 Methylmalonyl CoA, 326 recombinant DNA technology of, 518-521 Mature RNA, 458 Methylmalonyl CoA mutase, 210T, 326F Molecular disease, 53 Maxam, A., 194 Methylmalonyl CoA racemase, 326F Molecular genetics, 407 Maxam-Gilbert method, 194-196 N5-Methyltetrabydrofolate, 363 Molecular mass and weight, 48T Maximum velocity (V max), 89, 98 Methylthiocyanate, 52 Molybdenum, 346-347 Maxwell distribution, 88 Mevalonate, 338-340 dietary requirement, 21ST Mayer, R., 377 Mevalonate kinase, 338F Monellin, 128 McCarty, M., 185 Meyer, A., 377 Monera, 5 McClintock, B., 444 Meyerhof, 0., 241-242 1-Monoacylglycerol 3-phosphate, 337 Mediated transport: see Facilitated transport Micelles, 19-20 Monoacylglycerols, 143 Meister, A., 483 Michaelis, L., 92 , 208T , 355 Michaelis constant (Km), 97-100 Monocistronic mRNA, 471 Melting temperature (Tm), 191-192 magnitude, 1OOT Monod, J., 106,411,465 Membranes: see Biomembranes true and apparent, 101, I 03 Monoglycerides: see Monoacylglycerols Membrane transport, 159-163 Michaelis-Menten equation, 96-100 Monolayer (leaflet), 157-158 Menaquinone (vitamin~). 154 calculations, 98-99 Monomer, 54 Menten, M., 92 derivation,97-98 Monooxygenase,354-355 2-Mercaptoethanol, 42 Lineweaver-Burk transformations of, 99, Monosaccharides 13-Mercaptoethylarnine, 278 101, 103 biosynthesis, 268 6-Mercaptopurine, 368-369 Michaelis-Menten kinetics, 96-103 derived, 122-126 Meselson, M., 425, 479 Michel, H., 391 ring structures, 120--122 Meselson-Stabl experiment, 425--426 Micrococcus luteus, 441 See also Carbohydrates Meso compound, 509 Microfibril, 76, 131 Montagnier, L., 463 Mesophyll cells, 399--400 Microfilaments, 10 mRNA: see Messenger RNA INDEX 561

Mucopolysaccharide: see Glycosaminoglycans Negative superhelix, 184-185, 433F Nuclease S 1' !SOT Mueller-Hill, B., 467 Nemethy, G., 106 Nucleases, 179-180 Mulder, G., 47 Nernst equation, 295 Nucleic acids, 171-200 Multienzyme systems, 104,279, 283, 299, Nerve gas, 96 base pairing in, 181 301,330 Nerve impulse transmission, 164-165 digestion of, 213 Multifunctional enzymes, I 04, 332, 427 Net charge, estimation, 34-35 double helices, 183T Multiple sclerosis (MS ), 14 7 Neuberg, C., 241 hydrolysis of, 179-180 Murein: see Peptidoglycan Neurath, H., 69 noncovalent interactions in, 181-182 Muscle, metabolism in, 247F, 251, 265F Neurospora crassa, 185T sequencing of, 194-197 Mutagens, 439-440 peptides, 42 shorthand notations for, 176-177 Mutants, 212-213 Neutral fats: see Fats sizes, 177-179 Mutarotation, 120-122 NHI-proteins: see Nonheme-iron proteins structure, 176-177, 179-185 Mutase, 245 Niacin: see Nicotinic acid See also DNA, RNA Mutations, 420-421,439-440 Nick, 184 Nuclein, 171 Mycoplasma hominis, 178T Nicking-dosing enzyme, 184-185 Nucleoid: see Nuclear region Myelin, 147, 156T Nick translation, 428 Nucleolus, 9 Myocardial infarct, 351 Nicolson, G., 158 Nucleophile, 507 Myoglobin, 48T Nicotinamide, 275 Nucleoplasm, 454 isoelectric point, 80 Nicotinamide adenine dinucleotide: see N ucleoproteins, 178 oxygen binding by, 70-71 NAD+/NADH antibiotics, 17 4 unit evolutionary period of, 60F Nicotinamide adenine dinucleotide phosphate: Nucleoside diphosphate kinase, 208T, 261, X-ray diffraction of, 64-65 see NADP+ /NADPH 283,363 myo-inositol, 124 Nicotinamide coenzymes, 216T Nucleoside diphosphates Myosin, 77 Nicotinamide mononucleotide, 275, 435F as hexose carriers, 252, 260-261, 267-268 Myristic acid, 143 Nicotinic acid (niacin), 215T, 216T, 275 reduction of, 363-365 Niemann-Pick disease, 210T Nucleoside monophosphates, 175F NAD+/NADH (nicotinamide adenine Night blindness, 152 Nucleosides, 172-175 dinucleotide) Ninhydrin, 34 anti and syn conformations, 173-174 in alcoholic fermentation, 206 Nirenberg, M., 417-418 Nucleosomes, 188-189 in carbohydrate metabolism, 243-244, 250, Nirenberg-type experiment, 417-418, 481 Nucleotidase, 368-370 266,279 Nitrate assimilation, 346-347 Nucleotide-linked sugars, 252, 260-261, in citric acid cycle, 287 Nitrate reductase, 34 7 267-268 in DNA ligase reaction, 434-435 Nitrite reductase, 347 Nucleotide maps, 194 in electron transport system, 299-301 Nitrogenase,346-347 Nucleotides, 175-176 in glyoxylate cycle, 288-289 Nitrogen balance, 349 catabolism, 368-371 interconversion of NAD+ and NADP+, 397 Nitrogen bases: see Purines, Pyrimidines functions, 360 in lactate fermentation, 206 Nitrogen cycle, 346F Nucleotidyl transferase, 458 in lipid metabolism, 320, 324, 335, 337, Nitrogen equilibrium, 349 Nucleus, 8-9 341 Nitrogen excretion, 359-360 Nutrasweet: see Aspartame mitochondrial transport of, 310-312 Nitrogen fixation, 345-347 Nutrition, human, 213-217 in nitrogen metabolism, 347-348, 352, p-Nitrophenol, 109 caloric value of foods, 50, 117, 141 366F p-Nitrophenyl acetate, 109 recommended dietary allowances (RDAs), oxidation-reduction of, 275-276 Nitrous acid mutants, 450 215T reduction potential, 294 T N-linked oligosaccharides: see Oligosaccha- required nutrients, 21ST in tricarboxylate transport system, 330F rides Nutritional mutants: see Auxotroph ultraviolet absorbance, 276F NMN: see Nicotinamide mononucleotide NADH-CoQ reductase, 302T Noncompetitive inhibition, 101-103 Ochoa, S., 274 NADP+ /NADPH (nicotinamide adenine Noncovalent interactions, 18-21 Ochre codon, 416 dinucleotide phosphate) Noncyclic photophosphorylation, 389 Ogston, A., 86 in Hatch-Slack pathway, 400 Non essential amino acids, 31 Oils, 144 interconversion of NADP+ and NAD+, 397 Nonheme-iron proteins (NHI-proteins), 296, Okazaki, R., 434 in lipid metabolism, 329, 333-334, 298 Okazaki fragments, 434 338-341 Non-mediated transport: see Passive transport Oleic acid, 143-144 in nitrogen metabolism, 347-348 Non-overlapping code, 416 Oligomer, 54 in nucleotide metabolism, 363-365 Nonreducing end, oligosaccharides, 127 Oligomycin, 307 oxidation-reduction of, 275-276 Nonsaponifiable lipids, 145 Oligonucleotide, 176 in phosphate pathway, 253-255 Nonsense codons: see Termination codons Oligopeptide, 35 in photosynthesis, 384-385, 387-397 Nonsense mutations, 421 Oligosaccharides, 126-128 reduction potential, 294 T Norepinephrine, 209T biosynthesis, 268 in tricarboxylate transport system, 330F Northrop, J., 84 N-and 0-linked 133, 268, 497 ultraviolet absorbance, 276F N-terminus: see Amino terminus See also Cellobiose Na+ -K+ ATPase, 162-163 Nuclear envelope, 8 0-linked oligosaccharides: see Oligosaccha• Negative-strand viruses, 446-447 Nuclear region (nucleoid), 8 rides 562 INDEX

OMP: see Orotidine monophosphate Oxygen (cont.) Peptidyl transferase, 490-493 Oncogenes, 464 reduction by cytochrome oxidase, 299-302 Peptidyl-tRNA, 479-480 Oncogenic viruses, 460, 462 toxicity of partial! y reduced, 308-309 Perfusion, 211 One-carbon fragment metabolism, 361 Oxygen debt, 247 Perhydrocyclopentanophenanthrene, 155 Opal codon, 416 Oxygen saturation curve Periodate oxidation, 139 Operator, 465 effect of 2,3-bisphosphoglycerate, 75-76 Peripheral proteins, 158 Operon,465 of hemoglobin and myoglobin, 70-71, 74F, Periplasmic space, 135 Operon hypothesis, 465-467 76F Permease, 160 Opiates, 42 Oxyhemoglobin, 73F Pernicious anemia: see Anemia Opioid peptides, 42 Oxytocin, 40, 209T Peroxidase, 309 Opsin, 152-153 Peroxidation, 154 Optical rotation, 60, 190, 508-509 P700, P680: see Photosystems I and II Peroxisomes, 10, 397-398 Optimum pH and temperature, 92-94 Packaging of DNA, 187-189 Persoz, J. F., 83 Organelles, 9T PAGE: see Polyacrylamide gel electrophoresis Perutz, M., 64 OriC, 431 Palindromes, 188T, 193 PEST sequence, 498 Orientation effect, 21, 89 Palmitic acid, 143 pH, 13-15 Origin of life, 3-7, 27, 460 Palmito1eic acid, 143 calculations, 503 Origin of replication, 431 Palmitoyl-ACP, 334 gradient, 305-307, 386--387 Ornithine, 41, 356--359 Palmitoyl-ACP thioesterase, 332, 334 of human fluids, 14T Ornithine decarboxylase, 497 Palrnitoyl CoA, 337 Phage Ornithine transcarbamoy1ase, 358F Pancreas, 209T ~X174, 186,416-417,446 Orotate, 365-366 Pancreatic fluid, 213 DNA amount, 185T Orotate phosphoribosyl transferase, 366F Pantothenic acid, 216T, 278 DNA size, 178 Orotidine monophosphate (OMP), 366 dietary requirement, 215T T2/T4/T6, 186,441 Orotidylate decarboxylase, 366F Paper chromatography, 513 Phenazine methosulfate, 303F Orphan virus, 446 Paracrine hormones: see Hormones Phenylacetic acid, 320 Osmotic pressure, 50 Parnas, J ., 241 Phenylaceturic acid, 320 Osteomalacia: see Rickets Partition chromatography, 513 Phenylalanine, 28 Ovaries, 156 Passage: see Pore, Channels catabolism, 354-355 Overlap method, 56, 194 Passenger, recombinant DNA technology, 519 pK~ values, 29T Overlapping code, 416 Passive (non-mediated) transport, 159-160 Phenylalanine hydroxylase, 210T, 354-355 Overlapping genes, 416-417 Pasteur, L., XXV, 83, 249 Phenylalanine tolerance curve, 374 Overwinding, 184 Pasteur effect, 249-250 Phenylisothiocyanate, 39 Oxaloacetate, 264, 266, 280, 330F, 359, 400F Pasteur-Liebig controversy, 83 Phenylketonuria (PKU), 210T, 354-355 in anaplerotic reactions, 287 Pathways: see Metabolic pathways Phenyllactate, 354 in citric acid cycle, 281, 284 Pauling, L., 59 Phenylpyruvate, 354 in glyoxylate cycle, 288 Pauling scale, 11 Phenylthiohydantoin (PTH) amino acid, 39 in malate-aspartate shuttle, 311 F Payen, A., 83 Pheophytin,386 in transamination, 351-352 PCR: see Po1merase chain reaction Phosphamic acid, 228 in tricarboxylate transport system, 329-330 Pectin, 134 Phosphatases, 195, 262F, 336--337 Oxalosuccinate, 280 Pellagra, 216T Phosphate Oxidant, 524 Penicillin, 135 buffer system, 17 Oxidases: see Amino acid oxidase, Cy- Penicillium notatum, 135 as detergent builder, 146 tochrome oxidase Pentose, 119 Phosphatidate phosphatase, 337F 13-0xidation: see Beta oxidation Pentose phosphate pathway, 253-256 Phosphatidic acid, 146 Oxidation number, 373 carbon skeleton transformations in, 256F Phosphatidylcholine, 147 Oxidation-reduction, 523-526: see also light inactivation in, 396--397 Phosphatidyl ethanolamine, 147 Reduction potentials PEP carboxylase: see Phosphoenolpyruvate Phosphatidyl inositol, 147 Oxidative dearnination, 351-352 carboxylase Phosphatidylserine,147 Oxidative decarboxylation, 279 Pepsin, 213 Phosphocreatine,229 Oxidative phosphorylation, 208T, 305-312 kinetic parameters, 89T, lOOT Phosphodiesterases, 180T ATP synthase in, 302F, 307 specificity, 52T 3 ',5 '-Phosphodiester bond, 176--177 chemiosmotic coupling hypothesis of, Peptide bond, 35-36, 48 Phosphoenolpyruvate, 240, 245-246, 264, 305-307 configuration,36,59,62 399-400 controlot307-308 formation, 490-492 Phosphoenolpyruvate carboxykinase, 264-265 uncoupling of, 308 Peptide hormones, 40T, 209T Phosphoenolpyruvate carboxylase (PEP car- Oxidizing agents, 42, 308-309 Peptide maps, 52-53 boxylase), 399-400, 497 Oxidizing atmosphere, 4 Peptides, 27 Phosphofructokinase, 48T, 242, 397 &, 87T as antibiotics, 41, 160-161, 308 Phosphoglucoisomerase, 242, 253F Oxygen,385-386,397-399 naturally occurring, 40-43 Phosphoglucomutase, 252, 257, 260 binding to hemoglobin and myoglobin, Peptidoglycan (murein), 134-136, 135 6-Phosphogluconate, 254 70-76 Peptidyl site (P site), 478-479 6-Phosphogluconate dehydrogenase, 253-254 INDEX 563

6-Phosphogluconolactonase, 253-254 Phycobilin, 382-383 Polyuridylic acid (poly U), 417

6-Phosphogluconolactone, 254 Phylloquinone (vitamin K1), 154, 384 P/0 ratio (P/2e- ratio), 305 2-Phosphoglycerate, 240 Phytol, 382 Pore, 161 3-Phosphoglycerate, 240, 244-245, 392-398 pi: see Isoelectric point Positive-strand viruses, 446--447 Phosphoglycerate kinase, 244-245, 394F Pigments Positive superhelix, 184-185, 433F Phosphoglycerides: see Glycerophospholipids photosynthetic, 382-383 Postreplicative processing, 437 Phosphoglyceromutase, 245 skin, 355 Posttranscriptional processing Phosphoglycolate, 397-398 Pili, 9 of mRNA, 458--460 Phospholipases, 318 Piperazine-N,N' -bis[2-ethanesulfonic acid] ofrRNA, 458 Phospholipids, 146-147, 158 (PIPES), 17 oftRNA, 458 biosynthesis, 337-338 Pitch: see Helix pitch Posttranslational processing, 421, 496 in lipoproteins, 149T Pituitary gland, 209T, 219 Potential: see Action potential, reduction Phosphomannoisomerase, 252 pK~, 14-16 potential, transfer potential 5-Phosphomevalonate, 338 of amino acids, 29T Potential aldehyde group, 120 Phosphomevalonate kinase, 338F of Brpnsted acids, 16T Pox virus, 460 Phosphopantetheine group, 330-331 effects of variables on, 15-16, 33 Precursor, 212 Phosphopentose epimerase, 253F of purines and pyrimidines, 173F Precursor RNA (pre-RNA), 458 Phosphopentose isomerase, 253F PKU: see Phenylketonuria Prenyl transferase, 339F Phosphoprotein phosphatase, 261 F Planck's constant, 379 Pre-priming proteins, 436 5-Phosphoribosylarrrine,362 Planck's law, 379 Pribnow box, 455 Phosphoribosyl anthranilate isomerase, 468F Plants Priestley, J., 376 Phosphoribosyl pyrophosphate (PRPP), c3 and c4 types, 399--401 Primaquine, 255 361-364,362,366F cell wall of, 134 Primary structure Phosphoribosyl transferase, 363 Plaques, arterial, 165 as evolutionary index, 58-60 Phosphoribulose kinase, 394F Plasma (cell) membrane: see Biomembranes as molecular determinant, 56-58 Phosphoric acid, ionization and pK~ values, 14 Plasmids, 188, 520 of nucleic acids, 176-180 Phosphorolysis, 256-257, 368-369 Plastids, 189 of proteins, 51-58 Phosphorus, dietary requirement, 21ST Plastocyanin, 386-387 , 458 Phosphorylase: see Glycogen phosphorylase, Plastoquinones, 386-387 Primase, 435--436 Starch phosphorylase, Purine nucleoside Pleated sheet: see Beta pleated sheet Primer (initiator), 409 phosphorylase, Pyrimidine nucleoside Plectonemic coiling, 180 in DNA synthesis, 435--436 phosphorylase Pneumococcus, 186 in glycogenesis, 260-261 Phosphorylase kinase, 258-259 Point mutations, 420 Primordial soup, 4 Phosphorylation Poisoning, heavy metal ions, 67 Primosome, 436 oxidative, 305-312 Pol: see DNA polymerase Principle of unity and diversity, xxvi-xxvii photosynthetic, 386-387, 390-391 Polar reactions, 507 Procarboxypeptidase, I 03 substrate-level, 245-246, 283 Polio virus, 179T Processing, 409: see also Cotranscriptional Phosphoryl group, 125, 241 Polyacrylamide gel electrophoresis (PAGE), processing, Postreplicative processing, Phosphoserine, 30 195,197,517-518 Posttranscriptional processing, Posttrans• Photolyase, 440 Polyadenylation, 460--461 lational processing Photolysis, 383, 385-386 Polyaffinity theory, 85-86, 282 Processivity, 428--429 Photons, energy of, 378-379 Poly( A) polymerase, 460 Prochiral carbon, 86 Photooxidation Poly( A) tail, 459--460 Proenzymes, 103 of chlorophyll, 384 Polycistronic mRNA, 467 Prohormone, 152 of water, 385-386 Polyglutamic acid helix, 62 Prokaryotes Photophosphorylation, 386-387, 390-391 Polylysine helix, 62 cell wall of, 134-137 Photoreduction, 384-385 Polymerase chain reaction (PCR), 520-521 comparison with eukaryotes, 9-10 Photorespiration, 397-399 Polynucleotide kinase, 195 DNA of, 178T, 185T, 187-188 Photosynthesis, 375--404 Polynucleotide phosphorylase, 417 DNA replication, 431--438 dark reactions (Calvin cycle), 377, 392-397 Polynucleotides, 176 gene regulation, 464--4 70 energetics, 378-380, 387-390 Polyoma virus, 178T protein synthesis, 483--496 evolution of, 378 Polypeptide chain, 35-36, 60-62 ribosomes of, 412T historical review, 376-378 Polypeptide hormones, 40T, 209T Prolactin (PRL), 209T light reactions, 377, 384-391 Polypeptides, 35 , 28 in prokaryotes and eukaryotes, 378T, Polyribosome: see Polysome as helix breaking amino acid, 60 380-381 Polysaccharides pK~ values, 29T Z-scheme of, 385F biosynthesis, 267-268 in reverse turns, 63 Photosynthetic phosphorylation: see digestion of, 213-214 Promoter, 454--455 Photophosphorylation storage, 128-130 Proofreading Photosystems I and II (PSI, PSII), 383-384, structural, 130-131 in DNA replication, 428--430 389-391 Polysome (polyribosome), 480, 482F in translation, 485 Phototrophs, 8 Polyunsaturated fatty acids, 143 Propionyl CoA, 326 564 INDEX

Propionyl CoA carboxylase, 326F Proton pumps, 301, 306, 386 Pyruvate kinase, 246 Prostaglandins, 150, 209T Proto-oncogenes,464 Pyruvate phosphate dikinase, 400 Prosthetic group, 49, 94-95 Protoporphyrin IX, 71

Proteases, 52T: see also Serine proteases Proximity effect, 89 Q 10: see Temperature coefficient Protein binding, 70-76 PRPP: see Phosphoribosyl pyrophosphate Q/QH2 : see Coenzyme Q Protein coat, 178 PRPP synthase, 361-362 Q cycle, 306 Protein kinase, 258-259, 261-262, 336F PSI, PSII: see Photosystems I and II Quantum, 378-379 Proteinoids, 4 Pseudouridine, 414-415 Quantum yield, 377F, 380 Proteins, 47-82 Psicose, 119 Quaternary structure, 67 classification, 49-50 P site: see Peptidyl site Quinonoid dihydrobiopterin, 355 degradation, 497-498 Pulse-chase experiment, 474 Quinonoid intermediate, 351 denaturation, 69-70 Pump: see Active transport, Proton pumps dietary requirement, 215T Purification, in enzyme isolation, 90T digestion of, 213-214 Purine nucleoside phosphorylase, 368, 370 Racemic mixture, 508 domains in, 67 Purine nucleotides Radioactive isotopes, 211-212, 518 energy value of, 50 interconversions, 361, 363-365 Radioautography: see Autoradiography folding, 496 salvage pathways, 361, 363 Ramachandran plot, 62 globular and fibrous, 50, 65--66 Purines, 171-175 Random coil, 69-70, 190 half-life, 497-498 absorption spectra, 174F Raskas, H. J., 479 helical content, 65T acid-base properties, 173F Rate, 91, 222 isolation, 50-51 biosynthesis, 360-365 Rate constant, 96 molecular weights, 48T catabolism, 368-371 Rate-determining step, 97 number per cell, 470 Puromycin, 495 Rate equation, 96 primary structure, 51-58 Pyran, 122 RDA: see Recommended dietary allowance quaternary structure, 67 Pyranoside, 122 Reaction center, 383, 390F reverse turns in, 62--63 Pyridine-linked dehydrogenases, 275-276 Reaction order, 96 secondary structure, 58-63 Pyridine nucleotide coenzymes, 275 Reading franie, 416-417 sequencing of, 53-56 Pyridoxal, 350 Receptor-mediated endocytosis, 165-166 , 67-69 , 216T, 350 Receptors, 208, 258 supersecondary structure, 63--64 Pyridoxamine, 350 Reciprocal regulation, 262-263, 266-267 tertiary structure, 63-67 Pyridoxine, 350 Recombinant DNA technology, 518-521 X-ray analysis of, 64-65 Pyrimidine dimer: see dimer Recombination: see Genetic recombination See also Specific proteins Pyrimidine nucleoside phosphorylase, 371 Recombination repair, 442-443 Protein sequenator, 40 Pyrimidine nucleotides Recommended dietary allowance (RDA), Protein synthesis, 477-496 interconversions, 366-367 214-215 amino acid activation for, 483-485 salvage pathways, 367 Red blood cells: see Erythrocytes, Reticulo- coupling with transcription, 469-470, Pyrimidines, 171-175 cytes 482-483 absorption spectra, 174F Red drop, 377 elongation cycle of, 489-492 acid-base properties, 173F Redox potentials: see Reduction potentials energetics, 493-494 biosynthesis, 365-369 Reducing atmosphere, 4 in eukaryotes, 489 catabolism, 370-371 Reducing ends, oligosaccharides, 127 fidelity of, 485, 490 Pyroglutamic acid, 40 Reducing power: see NAD+fNADH, inhibition by antibiotics, 494-496 Pyrophosphatase, 260 NADP+ /NADPH initiation of, 486-489 Pyrophosphate (PPi), 176 Reducing sugars, 125, 127 overview, 410, 477-478 5-Pyrophosphomevalonate, 338 Reductant, 524 in prokaryotes, 483-496 Pyrophosphomevalonate decarboxylase, 338F Reduction, uphill and downhill, 384-385 rates of, 4 77 Pyrrolidine ring, 30 Reduction potentials, 293-295 regulation, 493 Pyruvate actual conditions, 294-295 termination of, 492-493 in amino acid metabolism, 351-353 in electron transport system, 302T, 304T Protein targeting, 496-497 in carbohydrate metabolism, 246-248, of half-reactions, 294T , 133-134 264-265 standard conditions, 293-294 Proteolipids, 149-150 in fermentation, 206 Reductive pentose phosphate cycle: see Calvin Proteolytic enzymes, 52T: see also Serine in Hatch-Slack pathway, 400F cycle proteases membrane transport of, 242, 329-330 Regulatory enzymes, 105-108 Prothrombin, 154-155 metabolic fates of, 248 allosteric, 106-108 Protista, 5 pK~ value, 16T covalently modified, 106 Protocells, 5 as source of acetyl CoA, 278-279, 281F Regulatory gene, 465 Protofibril, 76 in transamination, 352F Relaxed DNA, 184 Protomers, 107 Pyruvate carboxylase, 264, 287 Release factors (RF), 488T, 492-493 Proton acceptors and donors, 18 Pyruvate decarboxylase, 248 Renaturation, 70, 192

Proton gradient, 305-307, 386-387 Pyruvate dehydrogenase (E1), 279 Reovirus, 446 Proton-pumping ATPase: see ATP synthase Pyruvate dehydrogenase complex, 279, 281F Repetitive DNA, 470-471 INDEX 565

Replicase: see RNA replicase Ribosomes, 4ll-413 Sangerreaction,36-37 Replication: see DNA replication prokaryotic and eukaryotic, 412T Sanger reagent: see l -fluoro-2,4-dinitroben- Replication forks, 430-432 in protein synthesis, 478-480 zene Replicative form (RF), 446 Ribothymidine, 174,414 Saponifiable lipid, 145 Replicon, 431 ,5, 179,460-461 Saponification, 144 Replisome, 436 Ribulose, 119 Saponification number, 144-145 Repressible enzymes, 465 Ribulose 1,5-bisphosphate, 392-398 Satellite DNA, 470 Repressor, 465-466: see also Transcription Ribulose 1,5-bisphosphate carboxylase Saturation factors (rubisco), 392-398 in enzyme reactions, 92 Reptiles, nitrogen excretion, 360 Ribulose 5-phosphate, 254, 394F in membrane transport, 162-163 Resonance energy transfer, 379-380, 383 Ribulose 5-phosphate epimerase, 394F in oxygen binding, 70-71 Resonance hybrid, 35 Ribulose 5-phosphate isomerase, 394F Saturation curve: see Oxygen saturation curve Resonance stabilization, 229 Rich,A.,183 Schiff base, 350-351 Respiration, aerobic cellular, 226, 285 Ricin,49T Schleiden, M., xxv Respiratory chain: see Electron transport Rickets, 153-154 Schoenheimer, R., 339 system Rifampicin, 474 Schwann, T., xxv Respiratory complexes, 284,301-302 Right-handed helix, 60, 180 scRNP: see Small cytosolic ribonucleoproteins Respiratory control 307 RNA Scurvy, 124, 216T Respiratory quotient, 211 biosynthesis: see Transcription SDS: see Sodium dodecyl sulfate Restriction endonucleases, 193-194 primary structure, 17 6-177, 179-180 SDS-PAGE: see Sodium dodecyl sulfate poly• Reticulocytes, 480 secondary structure, 184, 412--414 acrylamide gel electrophoresis !l-eis-Retinal, 152 self-splicing, 460-461 Secondary structure all-trans-Retinal, 152 size, 179T ofDNA, 180-184 Retinal isomerase, 152F tertiary structure, 414 of proteins, 58-63 : see Vitamin A1 viral, 446-448 oftRNA, 184,413 Retinol dehydrogenase, 152F See also Messenger RNA, Ribosomal RNA, Second messenger, 208 Retinol equivalent, 21ST Small RNA, Transfer RNA Sedimentation coefficient, 515 Retrotransposon, 445 RNA-dependent DNA polymerase: see Sedoheptulose 1,7-bisphosphatase, 394F Retroviruses, 462 Reverse transcriptase Sedoheptulose 1,7-bisphosphate, 394F Reverse transcriptase, 460, 462 RNA-dependent RNA polymerase, 446-448 Sedoheptulose ?-phosphate, 255, 394F Reverse turns, 62-63 RNA-DNAhybrids, 193 Selenium, dietary requirement, 21ST RF: see Release factors, Replicative form RNA polymerases, 408-409, 453-454 Self-priming enzyme, 453 Rr value, 513 RNA replicase, 446-448 Self-splicing RNA, 460-461 Rho (p), 456-458 RNA viruses, classes, 446-447 Semiconservative replication, 425--426 Rhodopseudomonas viridis, 390-391 Robertson, J. D., 157 Senebier, J., 377 Rhodopsin, 152-153 Robison, R., 241 Sense codons, 416 Rh system, 137 Rod cells, 152 Sense (coding) strand, 408F, 410 Ribitol, 277 Rolling circle replication, 446-447 Sequence homology, 58 Riboflavin (vitamin B2), 21ST, 216T, 276- Rose, W. C., 27, 349 Sequencing 277 Rotational diffusion, 158-159 of nucleic acids, 194-197 Ribonuclease A, 180T Rotenone, 303F of proteins, 39-40, 53-56 Ribonuclease H, 462 Rough endoplasmic reticulum (RER), 9 Sequential model, 106-108 Ribonuclease P, 179, 458 Rous sarcoma virus, 460 Serine,28, 108-109, 146,337,398F Ribonucleases, 65T, 179-180 rRNA: see Ribosomal RNA pK~ values, 29T Ribonuclease T 1, 180T R,S-system, 510 Serine proteases, 103, 108-112, 164-165 Ribonucleic acid: see RNA R-type cells: see Pneumococcus Serotonin (5-hydroxytryptamine), 355-356 Ribonucleoside 5' -, from salvage, Ruben, S., 377 Serum albumin, 210T, 317 363 Rubisco: see Ribulose 1,5-bisphosphate Serum glutamate-oxaloacetate transaminase Ribonucleosides: see Nucleosides carboxylase (SGOT), 351 Ribonucleotide reductase, 363-365 Serum glutamate-pyruvate transaminase Ribonucleotides: see Nucleotides Sabatini, D., 496 (SGPT), 351 , 118, 122 Saccharin, 128 Sex hormones, 156 fermentability, 125T SAICAR: see Aminoimidazole succinylcar- SGOT test, 351 Ribose 1-phosphate, 368F, 370F boxamide ribonucleotide SGPT test, 351 Ribose 5-phosphate, 126, 394F SAICAR lyase, 362F Shine-Dalgarno sequence, 486-487 Ribosomal proteins (r-prot), 412 SAICAR synthetase, 362F Shuttle systems, 310-312 Ribosomal RNA (rRNA), 411-413 Saliva,213 Sialic acid (N-acetylneuraminic acid), 124, 147 amount per cell, 413 Salting in and salting out, 69 Sickle cell anemia: see Anemia processing of, 458 Salvage pathways, 361, 363, 367 Sigma cycle, 454 size, lOT, 179T SAM: see S-adenosylmethionine Sigma (a) subunit, 454 Ribosome binding assay, 418 Sanger, F., 36, 53, 194 Sigmoidal (S-shaped) binding curve, 70-71, Ribosome cycle, 479-481 Sanger-Coulson method, 196-197 107 566 INDEX

Signal hypothesis, 496--497 Split genes: see Discontinuous genes Sugar alcohols, 124 Signal peptidase, 497 Squalene, 339-340 Sugars: see Carbohydrates Signal peptide, 496 Squalene cyclase, 340 Suicide substrate, 84, 282, 369 Signal recognition particle (SRP), 496 Squalene epoxide, 340 Sulfa drugs, 102 Silencer, 4 71 Squalene monooxygenase, 340 Sulfanilamide, 102 Silent mutations, 421 Squalene synthase, 339F Sulfur amino acids: see Methionine, Cysteine Silk fibroin, 65 SRP: see Signal recognition particle Sulfur bacteria, 378, 381 Simple lipids, 141-146 SSB: see Single-strand binding protein Sulfur proteins: see Iron-sulfur proteins Simple proteins, 49 S-shaped (sigmoidal) binding curve, 70--71, Sumner, J. B., 83 Simple sugars: see Monosaccharides 107 Supercoil (superhelix), 184-185 Singer, J., 158 Stacking interactions, in DNA, 181-182 Superoxide anion radical, 42, 154, 308-309 Single-strand binding protein (SSB), 433, Staggered cuts, 193 Superoxide dismutase, 309 436T Stahl, F., 425 Supersecondary structures, 63-64 Sinsheimer, R. L., 186 Standard free energy change: see Free energy Svedberg equation, 515 Skin change Svedberg unit, 515 pigmentation of, 355 Standard hydrogen electrode, 294 SV virus, 178T in vitamin D formation, 153 Standard reduction potential: see Reduction Sweetness, 128T Slack, R., 399 potential Symport, 163 Small cytosolic ribonucleoproteins (scRNP), Standard states, 222-223 Synapse, 164 415 Staphylococcus aureus, 136F Synaptic cleft, 164 Small nuclear ribonucleoprotein (snRNP), Starch, 128-130,267 Syn conformation, 173-174 415,459 degradation, 129-130 Synonym codons, 416 Small nuclear RNA (snRNA), 415, 459 digestion of, 213-214 Synthetase recognition site, 484 Small RNA, 415 fermentability, 125T Szent-Gyiirgyi, A., 273 Smooth endoplasmic reticulum (SER), 9 Starch phosphorylase, 256 sn: see Stereospecific numbering Start codon, 486 Tagatose, 119 snRNA: see Small nuclear RNA Steady state, 96 Talose, 118 snRNP: see Small nuclear ribonucleoprotein Stearic acid, 142-143 Tangier's disease, 210T Soaps, 19-20, 144-146 Stem and loop: see Hairpin Targeting: see Protein targeting Sodium bicarbonate, 329 Stereoisomers, 509 TATA box, 455 Sodium dodecyl sulfate (SDS), 70 Stereospecific numbering (sn), 146 Taurine, 156 Sodium dodecyl sulfate polyacrylamide gel hormones, 209T Tautomerism, 172-173, 351F electrophoresis (SDS-PAGE), 517-518 Steroids, 155-156 Tay-Sachs disease, 210T

Sodium-potassium pump (Na+ -K+ ATPase), Sterols, 155 Tay-Sachs ganglioside (GM2), 148 162-163 Stop codons: see Termination codons TCA cycle: see Citric acid cycle Soft soaps, 144 Strand, 176 T cells, 463 Solubility, 11, 19-20, 67-69 Streptomyces, 174, 307 Temin, H., 460

Soluble RNA: see Transfer RNA Streptomycin, 495T Temperature coefficient (Q 10), 94 Somatotropin, 209T Stroma, 381 Temperature effects Sonication, 50 Stroma lamellae, 381 on DNA, 189-192 Sorbitol, 124 Structural gene, 465 on enzyme activity, 93-94 Sorbose, 119 S-type cells: see Pneumococcus on equilibrium constants, 225 S!iirensen, S. P. L., 13 Substrate, 84 on free energy changes, 225 Soret band, 298F Substrate anchoring, 89 on protein stability, 69-70 SOS repair, 443 Substrate constant (K,), 100 Template, 409 SOS response, 443 Substrate-level phosphorylation, 245-246, 283 Template (anticoding) strand, 408F, 410 Spacer (linker) DNA, 189 Subunit, 54 Terminal deoxynucleotidyl transferase, Sparing mechanism, 345 Succinate, 283-284 519-520 Special pair, chlorophyll, 391 pathway of electron transport, 301 Termination codons, 416,492 Specific activity, 89 pK~ values, 16T Terminator form of mRNA, 469F Specific heat, 12 Succinate-CoQ reductase, 302T Terminator utilization substance (tus), 437 Specific interactions school, 420 Succinate dehydrogenase, 208T, 284 Terpenoids, 151 Specificity constant (kca/Km), 1OOT Succinate thiokinase (succinyl CoA synthase), Tertiary hydrogen bonds, 414 Specific rotation, 508 283-284 Tertiary structure Spectrophotometry, 511 Succinyl CoA, 283, 286, 326 ofDNA, 184-185 , synthesis, 337-338 Succinyl CoA synthase: see Succinate thio• of proteins, 63-67 Sphingomyelin, 148 kinase oftRNA, 414 Sphingomyelinase, 210T Sucrose, 127-128,268 Testes, 156 Sphingophospholipids 147 fermentability, 125T Testosterone, 156, 209T Sphingosine, 147 sweetness, 128T Tetracycline,495 Spliceosome, 459 Sucrose 6-phosphate, 268 Tetrahedral intermediate, 112 Splicing, 458-461, 519-520 Sugar acids, 122-124 Tetrahydrobiopterin, 355 INDEX 5&7

Tetrahydrofolate (THF), 361-363 Transaminases, 350-352 Triose-phosphate isomerase, 243, 253F, 394F Tetrahymena, 179, 460 Transamination, 350-352 Tripeptide, 35 Tetramethyl-p-phenylenediamine (TMPD), Transcarboxylase, 330-331 Triplet: see Codon 305,315 Transcription, 408, 453-475 Tris(hydroxymethyl)aminomethane (TRIS), Tetrose, 119 coupling with translation, 469-470, 17 Thermal denaturation profile, 190-192 482-483 Tristearin, 144 Thermoacidophiles, 7 elongation and termination in, 456-458 tRNA: see transfer RNA Thermolysin, specificity, 52T initiation of, 455-456 tRNA~et (tRNNMe<), 486 Thermophiles, 521 rate of, 474 trp operon, 468-470 Thermostable enzymes, 93 visualization of, 457F trp repressor, 468 Theta replication, 431 Transcriptional control, 464, 4 71-472 True fats: see Fats THF: see Tetrahydrofolate Transcription factors, 4 71 Trypsin, specificity, 52T Thiamine, 21ST, 216T, 278-279 Transduction, 444 Trypsinogen, I 03-104 Thiamine pyrophosphate (TPP), 216T, , 87T Tryptophan, 28 254-255,278-279 Transfer potentials, 227 pK: values, 29T Thiazole nucleus, 278-279 Transfer RNA (tRNA), 194, 413-415 Tryptophan synthase, 89T, 468F Thioesters, 278 amount per cell, 413 Turnover number, 89 Thiogalactoside transacetylase, 465 base sequence of alanine, 413 Turtles, nitrogen excretion, 360 Thiokinase, 321-322 cloverleaf model of, 184, 413 tus: see Terminator utilization substance Thiol, 508T cognate, 484 Tyrosinase, 210T Thio1ase (!3-ketothiolase), 324-325, 328F isoacceptor, 413 Tyrosine, 28 Thiolysis, 324 L-shaped structure of, 414F catabolism, 354-355 Thioredoxin, 363-364, 396 processing, 458 pK~ values, 29T Thioredoxin reductase, 364 size, 179T Tyrosyl tRNA synthetase, 89T Threonine, 28 Transformation, 443 optical isomers, 508 Transforming principle, 185-186 Ubiquinone: see Coenzyme Q pK~ values, 29T Transformylase, 486 Ubiquitin, 498 Threose, 118 Transglycosylase, 261 Ubiquitination, 498 Thromboxanes, 150-151 Transhydrogenase,397 UDP: see 5'-diphosphate Thylakoid disks, 381, 386-387 Transimination, 350-351 UDP-galactose, 252, 268 Thymidine, 367,370 Transition mutations, 420, 439 UDP-galactose epimerase, 252 Thymidine kinase, 367 Transition state: see Activated complex UDP-glucose, 252,260-261,267-268 Thymidine phosphorylase, 370F Transition state stabilization, 90 UDP-glucose pyrophosphorylase, 252, 260 , 366-367, 369 Transition state theory, 87-89 UEP: see Unit evolutionary period Thymine, 172 , 253-255, 394F Ultimate hormone, 208 pK~ values, 173F Translation: see Protein synthesis Ultracentrifugation, 515-516 Thymine dimers, 439-440 Translational control, 464 Ultraviolet absorbance, of DNA and RNA, repair mechanisms for, 441-443 (EF-G), 160, 492 190-192 Thymus nucleic acid, 171 Translocation, 492 UMP: see Uridine 5 '-monophosphate Thyroid, 209T Transmembrane protein, !58 Uncompetitive inhibition, IOIF, 103 Thyrotropin (TSH), 209T Transmittance, 511 Uncouplers, 308 Thyrotropin releasing factor (TRF), 40, 209T Transpeptidation, 492 Underwinding, 184

Thyroxine (T4 ), 209T Transport: see Membrane transport Unit evolutionary period (UEP), 58, 60F Tissue preparations, 211 Transport protein, 160 Unit membrane hypothesis, !57 Titrations, 15F,32-33 Transposable elements: see Mobile genetic Unity and diversity, principle of, xxvi-xxvii T m: see Melting temperature elements Unsaturated fatty acids Tobacco mosaic virus (TMV), 48T, 178- Transposase, 445 catabolism, 326-327 179 Transposition, 444-445 in lipid bilayers, !58 Tobacco necrosis virus, 48T Transposon, 445 Untranscribed DNA, 470 a-Tocopherol, 154 Transverse diffusion: see Flip-flop Unzippering, 192 a-Tocopherol equivalent, 21ST Transversion mutations, 420, 439 Uphill reactions, 206-207 Tollens' reagent, 125 Trehalose, 140 Uphill reductions, 384 Topoisomerase I: see Nicking-closing enzyme Triacylglycerols, 144, 337 Upstream, 454-455 Topoisomerase II: see DNA gyrase in lipoproteins, 149T , 172 Tortoises, nitrogen excretion, 360 See also Fats pK: values, 173F Tosyl-L-phenylalanyl chloromethyl ketone Tricarboxylate transport system, 329-330 Urea (TPCK), 109 Tricarboxylic acid cycle (TCA cycle): excretion,360,369-370 T2ff4tr6 phage: see Phage see Citric acid cycle as protein denaturant, 70 TPP: see Thiamine pyrophosphate Triglycerides: see Triacylglycerols Urea cycle, 356-359

Trace elements, 214 Triiodothyronine (T3 ), 209T committed step, 357 , 253-255 Triose, 119 energetics, 358-359 Transamidase,268 Triose kinase, 251 metabolic links of, 359F 568 INDEX

Urease, 83-84 Viruses (cont.) Watson-Crick base pairs, 181-182 isoelectric point, 80 nucleic acid replication, 446-448 Waxes, 143 kinetic parameters, 89T, lOOT nucleic acid size, 178T, 179T Weak interactions, 18 13-Ureidoisobutyrate, 370 pathogenicity of, 447-448, 460-463 Wobble base, 413F, 421 13-Ureidopropionate, 370 See also Phage Wobble hypothesis, 421 Ureotelic organisms, 360 Visual cycle, 1S2-1S3 Woehler, F., xxv Uric acid Vitalism, xxv Wyman, J., 106 catabolism, 368-371 , 1Sl-1S2, 21ST

excretion, 360 VitaminA1, !Sl-152 Xanthine, 368-370 Uricotelic organisms, 360 Vitamin Az, 152 , 368F, 370 Uridine, 370 Vitamin B 1: see Thiamine Xanthophylls, 383 Uridine 5' -diphosphate (UDP), 260-261 Vitamin B2: see Riboflavin Xanthosine, 368 Uridine diphosphate galactose: Vitamin B6 , 215T, 216T, 350 Xanthosine S' -monophosphate (XMP), 368 see UDP-galactose Vitamin B 12, 21ST, 216T, 326, 373 Xenopus laevis, 472 Uridine diphosphate glucose: Vitamin C: see Ascorbate Xeroderma pigmentosum, 441-442 see UDP-glucose Vitamin D, 1S2-1S4, 21ST Xerophthalmia, 1S2 Uridine 5' -monophosphate (UMP), 366--367, Vitamin 0 2: see Ergocalciferol X-ray diffraction, 48, 64-6S, 180 370-371 Vitamin 0 3 : see Cholecalciferol Xylose, 118 Uridine phosphorylase, 370F , 154, 21ST Xylulose, 119 Uridine 5 '-triphosphate (UTP), 260-261 , 1S4-1S5, 21ST Xylulose S-phosphate, 255, 394F

Uridylic acid: see Uridine S '-monophosphate Vitamin K 1: see Phylloquinone Uronic acid, 122 Vitamin Kz: see Menaquinone Yanofski, C., 469 Vitamins, 94-9S, 216-217 Yeast, 12ST, 178T, 248 Vacuoles, 10 definition, 216 Yeast nucleic acid, 171 Valine,28 fat-soluble, lSI-ISS Yield, in enzyme isolation, 90T pK~ values, 29T water-soluble, 94-9S, 216T Young, W.,239 Valinomycin, 160-161, 308 VLDL (very low-density lipoproteins), 149T Van der Waals interactions, 21, 18T V max: see Maximum velocity Z-DNA, 183-184 Vane, J., 150 Voltage-gated channel, 161 Zero order reaction, 96 Van Niel, C., 377 Zinc, dietary requirement, 21ST Van't Hoff equation, 22S Wang, A., 183 Zinc finger, 4 71 Vasopressin, 40, 209T Warburg, 0., 241 Zone electrophoresis, S17 Vector, recombinant DNA technology, S19 Water, 10-18 Z-scheme of photosynthesis, 38SF Vegetable oils, 144 hydrogen bonding in, 11-12 Zwitterion, 31 Velocity: see Rate ionization and ion product, 12-13 Zymase, 239 Very low-density lipoproteins: see VLDL photooxidation of, 384-386 Zymogens, 103 Viroid, 179 properties, 11-13 Viruses Water-soluble vitamins, 94-9S, 216T classes, 446-447 Watson, J., 180