Chapter 14 Essential Questions • Before This Class, Ask Your Self the Following Questions: – How Fast an Enzyme Could Accelerate a Reaction? Reginald H

Home , Chorismate mutase

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Chapter 14 Essential Questions • Before this class, ask your self the following questions: – How fast an enzyme could accelerate a reaction? Reginald H. Garrett Charles M. Grisham – Why enzyme could accelerate a reaction? Mechanisms of Enzyme Action – How enzymes could accelerate a reaction? – What are the universal chemical principles that Դৣ influence the mechanisms of enzymes and allow us to ᑣ٫݅ ፐำᆛઠ understand their enormous catalyyptic power? http://lms.ls.ntou.edu.tw/course/106 hji@[email protected] dtdu.tw

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Outline How Much Enzyme-Induced Rate Accelerations? • Part I: The general concepts of enzyme catalysis • TillTypically 107 – 1014 greater – What are the magnitudes of enzyme-induced rate accelerations? • Why? 2 reasons…. – What role does transition-state stabilization play in enzyme – Sta biliza tion o f tititttransition state catalysis? – Destabilization of ES – How does destabilization of ES affect enzyme catalysis? • due to strain, desolvation or electrostatic effects – How tightly do transition-state analogs bind to the active site? • How? 5 catalytic mechanisms…. • Part II: The mechanisms of catalysis – covalent catalysis • Part III: What can be learned from typical enzyme – general acid or base catalysis mechanisms? – Low-Barrier H-bonds – 3 examples: serine protease, aspartic protease and chorismate – metal ion catalysis mutase! – ppyroximity/orientation ( Near-Attack Conformation).

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14. 1 What Are the Magnitudes of Enzyme- What is transition state? Induced Rate Accelerations? • The structure represents , as nearly as possible, the transition between the reactants and products, and it is known as the transition state. • Transition =\= intermediates ( Ex: ES or EP) – Intermediates are longer-lived, with lifetimes in the range of 10-13 sec to 10-3 sec. – A typical transition state has very short lifetime, typically 10-13 sec.

- - H-O-H + Cl- H-O H Cl HO- + H-Cl

Transition state existed even there is no enzyme 5 6

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Reason 1: Transition-State Stabilization Reason 2: Destabilize ES complex

• With enzyme, the transition state changed from X‡ to The dissociation constant for the EX‡ enzyme –substrate complex ‡ Ks • Activation energy difference (G ) between ES and ES E+S ‡ ‡ EX is smaller than between S and X , therefore the [E] [S] Ks = catalyypzed rate of product formation will be faster. [ES]

No enzyme! Enzyme catalysis The dissociation constant for the transition-state complex K EX‡ T E+X‡ [E] [X‡] ‡ ‡ Gu >Ge KT = [EX‡] Enzyme catalysis requires To stabilize EX‡ more! KT< Ks b Dissociation of ES facilitates reaction ke/ku KS/KT 7 8 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture The binding of S to E must be favorable Destabilization of ES but not too favorable!

• ES cannot be "too tight" – The idea is to make Strain the energy barrier between ES and EX‡ small!

• Intrinsic binding energy Gb • Some amino acid of enzyme favor to bind substrate, making Desolvation Gb negative! • Compensation • Entropy loss due to the binding of E and S (T S) • Destabilization of ES ( Gd) by strain, distortion, desolvation , Distortion and similar effects.

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

No Destabilization of ES No Catalysis

• Net energy difference • RiiRaising the energy o fESif ES raises the ra t!te! between E + S and the ES complex is the sum of …. 1. intrinsic binding energy, Gb 2. the entropy loss on binding, -TS 3. the dis tor tion energy, . Gd

12 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture 14.4 How Tiggyhtly Do Transition-State First TSA case: Proline racemase Analogs Bind to the Active Site? • ThTo have b bttetter k/kke/kuvalKlue, K /K shldbbi!hould be big! • Proline racemase , a bacterial enzyme, catalyzes S T the interconversion of D and L-proline. –KT should be small – The affinity of the enzyme for the transition state may • The TSA, pyrrole-2-carboxylate, bound to the be 10 -20 to 10-26 M! enzyme 160 times more tightly than L-proline • Can we prove such kind of?f tight binding? • Transition state analogs (TSAs) – are stable molecules that are chemically and structurally similar to the transition state – bind more strongly than a substrate – transition-state analogs are ppyotent enzyme inhibitors 13 14

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

TSAs are also very strong inhibitors

(b) Pu rin e ri bosi de inhi bi ts aden osin e deamin ase. Th e Figure 14. 6 (a ) Phosp hog lyco lo hy droxamate is an ana log o f t he hydrated form is an analog of the transition state of the enediolate transition state of the yeast aldolase reaction. 15 reaction. 16 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Transition-State Analogs Make Our World EndofPart1End of Part 1 Better • Enzymes are often targets for drugs and • Ask yourself… other beneficial agents – What is transition state? • Transition state analogs often make ideal – What is intermediate? enzyme inhibitors – What is the two reasons that enzyme could – Enalapril and Aliskiren lower blood pressure accelerate reaction? – Statins lower serum cholesterol – Wha t e ffec ts cou ld des ta bilize ES comp lex ? – Protease inhibitors are AIDS drugs – What is TSA? What can TSA tell us? – Juvenile hormone esterase is a pesticide target – Tamiflu is a viral neuraminidase inhibitor 17 18

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

14.5 What Are the Mechanisms of Catalysis? Protein Motions Are Essential to Enzyme Catalysis • Protein motions are essential to enzyme • Proteins are constantly moving catalysis – bonds vibrate, side chains bend and rotate, backbone loops wiggle and sway, and whole domains move as a • 5 major mechanisms: unit – Enzymes facilitate formation of near-attack • Protein motions support catalysis in several ways. complexes Active site conformation changes can: – Covalent catalysis – Assist substrate binding – General acid-base catalysis – Bring catalytic groups into position – Low-barrier hydrogen bonds – IdInduce f ormati on of fNAC NACs – Assist in bond making and bond breaking – Metal ion catalysis – Facilit a te convers ion o f su bs tra te to pro duc t

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Example: Human cyclophilin A Evident of enzyme movement

NMR showed that several active-site residues undergo a prollilylisomerase, whic h greater motion during catalysis than residues elsewhere catalyzes the interconversion in the protein. btbetween trans and cis conformations of proline in peptides.

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Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

Some Review of General Chemistry How to read and write mechanisms Step 1 • The custom of writing chemical reaction by Gilbert Newton Lewis and Sir Robert • In written mechanisms, a curved arrow shows RbiRobinson the movement of an electron pair • Review of the concepts • And thus the movement of a pair of electrons – Lewis dot structures from a filled orbital to an empty one – Valence electrons and formal charge • A fllfull arrow hea d representtltis an electron pair – Formal charge = group number – nonbonding •A half arrowhead represents a single electron eltlectrons – (1/2 s hare d e lec trons ) – Electronegativity is also important: – F > O > N > C > H 23 24 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture How to read and write mechanisms How to read and write mechanisms Step 2 Step 3 For a bdbond-bkibreaking event, th e arrow beg ins in the m iddle o f • It h as been esti mat ed th at 75% o f the s teps in the bond, and the arrow points to the atom that will accept the enzyme reaction mechanisms are proton (H+) electrons. tftransfers. • If the proton is donated or accepted by a group on the enzyme, it is often convenient (and traditional) to represent the group as “B”, for For a bond-making event, the arrow begins at the source of “base”, even if B is protonated and behaving as the electrons (for example, a nonbonded pair), and the arrowhead points to the atom where the new bond will be an acid: formed.

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Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture How to read and write mechanisms How to read and write mechanisms Step 4 • It is important to appreciate that a proton transfer can change a nucleophile into an electrophile, and vice versa. • Thus, it is necessary to consider: – The protonation states of substrate and active-site residues An active-site histidine, which might normally be protonated, can – How pKa values can change in the be deprotonated by another group environment of the active site and then act as a base, accepting a proton from the substrate.

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How to read and write mechanisms Mechanism 1: Near-attack complexes

Water can often act as an acid or base at the active site • X-ray crystal structure studies have shown that the through proton transfer with an assisting active-site residue: reacting atoms and catalytic groups are precisely positioned for their roles. • The preorganization – selects substrate conformations – the reacting atoms are in van der Waals contact – at an angl e resembli ng the bon dtd to be forme did in the transition state • Thomas Bruice has termed such arrangements This type of chemistry is the basis for general acid-base near-attack conformations (NACs) catalyy(sis (discussed on pgpages 430-431). • NACs are precursors to reaction transition states

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Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

Enzymes facilitate formation of near- Requirements of the formation of near- attack complexes attack complexes

Figure 14.7 NACs are • In the absence of an enzyme, potential characterized as having reactant molecules adopt a NAC only reacting atoms within 3.2 Å and an approach angle of Ʋ15Ʊ about 0. 0001% of the time of the bonding angle in the • On the other hand, NACs have been transition state. shown to form in enzyme active sites from 1% to 70% of the time

31 32 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Enzymes facilitate formation of near- The active site of liver alcohol attack complexes dehydrogenase – a near-attack complex.

• The energy separation between the NAC and the transition state is approximately the same in the presence and absence of the enzyme. • In an enzyme active site, the NAC forms more readily than in the uncatalyzed • An ordered,,g single-displacement Rx. reaction. • Intermediate exists as a NAC 60% of the time 33 34

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Mechanism 2: Covalent Catalysis Mechanism of Covalent catalysis

• Some enzyme reactions derive much of • The side chains of amino acids in proteins their rate acceleration from the formation (Enzyme) offer a variety of nucleophilic centers of covalent bonds between enzyme and for catalysis, including amines, carboxylates, aryl and alkyl hydroxyls, imidazoles, and thiol substrate groups. • These groups readily attack electrophilic centers BX + Y BY + X of substrates, forming covalently bonded BX+EnzE:B+X+YEnz+BYBX + Enz E : B + X + Y Enz + BY enzyme-substrate intermediates. • Thecovalentintermediatecanbeattackedina second step by water or by a second substrate, covalent intermediate forming the desired product 35 36 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Enzymes that form covalent Nucleophilic groups in enzymes intermediates

Nucleophilic group Amino Acid OH Serine SH Cysteine COO- Aspartic acid

NH2 Lysine imidazole Histidine

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Example of Covalent Catalysis Mechanism 3: Acid - Base Catalysis

• There are two t ypes o f ac id-btlibase catalysis: (1) specific acid-base catalysis, in which H+ or OH- accelerates the reaction, (2) general acid-base catalysis, in which an acid or base other than H+ or OH- accelerates the reaction.

Typical electrophilic What are “acid and base” ? centers in substrates Acid Base ildinclude Arrhenius H+ forming OH- forming • phosphoryl group Bronsted-Lowry H+ donor H+ acceptor • acyl group LiLewis e- acceptor e- donor • glycosyl group 39 See General chemistry chap 15 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

Specific acid or base catalysis General acid-base catalysis

In specific acid or base catalysis, the • general acid-base catalysis is catalysis in which a buffer concentration has no effect. proton is transferred in the transition state.

Jo!tqfdjgjd!bdje.cbtf! In general acid - base catalysis, in dbubmztjt-!I, ps!PI. which an ionizable buffer may dpodfousb ujpo!b ggfd ut! uif! donate or accept a proton in the transition state, k is dependent sfbdujpo!sbuf-!lpct jt!qI. obs efqfoefou-!cvu!cvggfst! on buffer concentration. dpodfousbujpo!) xij d i!bddfqu! ps!epobuf!I,0PI.*!ibwf!op! fggfdu/

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Catalysis of p-nitrophenylacetate hydrolysis Mechanism 4: Low-Barrier Hydrogen Bonds can occur either by specific acid hydrolysis or (LBHBs) by ge ne ra l base catal ysi s • In normal hyygdrogen bonds (O:•••H–O), the O•••O separation is 2.8 Å •InLBHBs, the O•••O separation decreases until the H a tom becomes centered , l eavi ng th e H atom to freely exchange between the two O atoms •pKa values of the two electronegative atoms must be similar • LBHB are very strong, transient intermediates that helppy to accelerate enzyme-catalyzed reactions Histidine is most effective general acid or base because its - LBHB may be to redistribute electron density in the pKa is near 7! intermediate Ex: Serine Proteases, Asp Protease 43 44 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Low-Barrier Hydrogen Bonds (LBHBs) Mechanism 5: Metal-Ion Catalysis

• Many enzymes require metal ions for maximal activity. • metalloenzyme : enzyme binds the metal very tiggyhtly or requires the metal ion to maintain its stable, native state. (metal is coenzyme) - e.g. Fe, Cu, Zn, Mn, Co. • metal-activated enzymes :Enzymes that bind metal ions more weakly, perhaps only during the

O-H-O hydrogen bond of length 0.28 the O-O distances is the O-O distance is 0.23 to catalytic cycle. nm, with the hydrogen bond order for 0.25 nm 0.24 nm, and the bond order the st ronger O -HiH in terac tion is of each O-H interaction is - ege.g Na, K, Mg, Ca approximately 1.0, and the weaker O- 0.5. H interaction is 0.07. 45 46

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Example of Metal Ion Catalysis Functions of Metal-Ion

• One role for metals in metal-activated enzymes and metalloenzymes is to act as electrophilic catalysts, stabilizing the increased electron density or negative charge that can develop during reactions. • Another potential function: coordination and Figure 14.14 Thermolysin is an endoprotease with a catalytic increase the acidity of a nucleophile Zn2+ ion in the ac tive sit e. The Zn2+ ion sta bilizes the build up o f negative charge on the peptide carbonyl oxygen, as a glutamate residue deprotonates water, promoting hydroxide attack on the carbonyl carbon. 48 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture How Do Active -Site Residues Interact to EndofPart2End of Part 2 Support Catalysis? • About half of the amino acids engage • Ask yourself… directlyyy in catalytic effects in enz yme – What are general mechanism of enzyme active sites catltalys i?is? – Do you know how to read the reaction • Other residues may function in secondary mechanism? roles in the active site: – What are ggpgpeneral nucleophilic groups in – Raising or lowering catalytic residue pKa amino acid side chains? values – What’s different between specific and general – Orientation of catalytic residues acid-base reaction? – Charge stabilization – What are the functions of metal ions in – Proton transfers via hydrogen tunneling enzyme catalysis? 49 50

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Example 1: The Serine Proteases 14. 5 What Can Be Learned From Typical Family Enzyme Mechanisms? • All involve a serine in catalysis - thus the name • Serine proteases includes • Three typical enz ymes to ex plain the – Trypsin, chymotrypsin, elastase are digestive mechanisms: enzymes and are synthesized in the pancreas and – The serine protease: secreted into the digestive tract as inactive proenzymes, or zymogens. • covalent catalysis, general acid-base catalysis, substrate selectivity, LBHB – Thrombin is a crucial enzyme in the blood-clotting cascade – The aspartic protease: – Subtilisin is a bacterial protease • general acid-base catalyy,sis, LBHB – Plasmin breaks down the fibrin polymers of blood – Chorismate mutase: clots. •NAC – Tissue plasminogen activator (TPA) specifically cleaves the proenzyme plasminogen, yielding plasmin 51 52 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture zymogen Catalytic Triad

• Proenzyme or zymogen: is an inactive • SiSer is par tf"tltitid"fSHit of a "catalytic triad" of Ser, His, enzyypme precursor. Asp • A zymogen requires a biochemical change • Serine proteases are homologous, but (such as a hydrolysis reaction revealing locations of the three crucial residues differ somewhat the active site, or changing the conf)figuration to reveal the active site) for it • Enzymologists agree to number the triad always as His57, Asp102, Ser195 to become an active enzyme. • Feed forward reaction (positive feedback)!

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Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Similarity of 3 Serine Proteases The Catalytic Triad of the Serine Proteases

-EhilEach circle represents one amino acid. Figure 14.16 Structure of - Numbering is based on the chyyyp()motrypsin (white) in a sequence of complex with eglin C (blue chymotrypsinogen. ribbon structure), a target 57 -Filled circles indicate residues subtbstra te. His (d)(red) is that are identical in all three flanked by Asp102 (gold) and proteins. Ser195 (green). The catalytic -Disulfide bonds are indicated site is filled by a peptide in yellow. segggment of eglin. Note how 195 -The positions of the three close Ser is to the peptide catalytically important active- that would be cleaved in the site resid ues (His57 , Asp 102, reaction. and Ser195) are indicated. 55 56 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

The su bs trat e-bin ding pock e ts Serine Protease Binding Pockets are determine the substrate selectivity Adapted to Particular Substrates

trypsin chymotrypsin elastase

Bulky threonine and valine residues at the opening Positive charge Ex: arginine and lysine residues Hydrophobic residues

Negative charge 57 58

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Burst kinetics tell the mechanism of How to assay the activity of protease? serine protease • Serine Proteases Cleave Simple Organic Esters, such as p-Nitrophenylacetate

Release of this part give yellow color!

Which protease would cut it? 59 60 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

Serine Protease Mechanism The Serine Protease Mechanism in Detail 1/5

• A mixture of covalent Binding of a model and general acid-base catalysis substrate. –Asp102 functions only to orient His57 – His57 acts as a FtifthFormation of the general acid and covalent ES complex base involves general base –Ser195 forms a catalysis by His57 covalent bond with peptide to be Covalent bond cleaved formation turns a trigonal C into a tetrahedral C 61 62

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture The Serine Protease Mechanism in Detail 3/5 The Serine Protease Mechanism in Detail 2/5

The amino product departs, making room His57 stabilized by a for an entering water LBHB. molecule.

pKa is different, how LBHB formation? Nucleophilic attack by water is facilitated by His57, acting as a collapse of the tetrahedral general base. intermediate releases the first product.

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The Serine Protease Mechanism in Detail 4/5 The Serine Protease Mechanism in Detail 5/5

Collapse of the tetrahedral intermediate cleaves the covalent intermediate, releasing the second product.

CbCarboxy l pro duc t re lease completes the serine protease mechanism .

Figure 14.21 The chymotrypsin mechanism: At the completion of th e reacti on, the s ide ch a ins of th e cat al yti c t ri ad are restored to their original states. 65 66

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Transition-State Stabilization in the The “ oxyan ion ho le ” Serine Proteases • The chymotrypsin mechanism involves two tetrahedral oxyanion intermediates • These intermediates are stabilized by a pair of amide groups that is termed the “oxyanion hole” • The amide N-H groups of Ser195 and Gly193 provide primary stabilization of the tetrahedral oxyanion The oxyanion hole of chymotrypsin stabilizes the tetrahedral oxyyganion intermediate seen in the mechanism of Figure 14.21. 67 68 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

El2ThAtiPtExample 2: The Aspartic Proteases Aspartic proteases play many roles in humans • All involve two Asp residues at the active site • These two Asp residues work together as generalidl acid-btltbase catalysts • Most aspartic proteases have a tertiary structure consisting of two lobes (N -terminal and C-terminal) with approximate two-fold symmetry

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Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture The Aspartic Proteases AtiPtMhiAspartic Protease Mechanism Most aspartic proteases exhibit a two-lobed structure. Each lblobe con tibttributes one cat tltialytic aspart ttate t o th e acti ve sit e. • ElitidtitiEnzymologists said aspartic proteases is HIV-1 protease is a homodimeric enzyme, with each general acid-base catalysis subunit contributing a catalytic Asp residue. – show one relatively low pKa, and one relatively high Figure 14.22 Structures of (a) HIV-1 protease and (b) pK pepsin. Pepsin ’ ssN N-terminal half is shown in red; the a C-terminal half is shown in blue. – once thought to represent pKa values of the two aspartate residues • Structural Chemists said it is a LBHB catalysis – LBHB disperse electron density (electron tl)tunnel)

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A Mechanism for the Aspartic Proteases HIV-1 Protease

• HIV-1 protease cleaves the polyprotein prodfhHIVducts of the HIV genome • This is a remarkable imitation of mammalian aspartic proteases •HIV-1 protease is a homodimer - more genetically economical for the virus • Active site is two-fold symmetric

Figure 14.24 Mechanism for the aspartic proteases. LBHBs play a ro le in s ta tes E, ES, ET’, EQ’ , and EP’Q .

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Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Proteolytic cleavage pattern for the HIV Protease Inhibitors Block the Active Site of genome HIV-1 Protease

Figure1427HIVFigure 14.27 HIV-1 protease complexed with the inhibitor Crixivan (red) made by Merck. The “flaps” that cover the active site are ggyreen; the catalytic active site As p residues Figure 14.26 HIV mRNA provides the genetic information for are violet. synthesis of a polyprotein. Cleavage yields the active products.75 76 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture

Protease Inhibitors Give Life to AIDS Patients Protease Inhibitors Give Life to AIDS Patients Protease inhibitors as AIDS drugs • If the HIV-1 protease can be selectively inhibited, then new HIV particles cannot form • Several novel protease inhibitors are currently marketed as AIDS drugs • Many such inhibitors work in a culture dish • However, a successful drug must be able to kill the virus in a human subject without blocking other essenti al prot eases i n th e b od y

Protease inhibitor drugs used by AIDS Patients 77 78

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Example 3: Chorismate Mutase Chorismate Mutase: A Model for Understanding Catalytic Power and Efficiency • Biosynthesis of Phe and Tyr in microbe and plant. • Single substrate! • ItIntramo lecul ar rearrangement • Good ex am pl e of th e catal yti c pow er of enzyme – Uncatalyzed reaction use the same transition state! Chorismate Intramolecular rearrangement Prephenate

79 80 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Chorismate Mutase: A Model for Understanding Two possible mechanisms of chorismate Catalytic Power and Efficiency rearrangement reaction

Figure 14.29 The Figure 24.28 A c l assi c Cl ai sen r earr an gem en t. Conv er si on of critical H atoms allyl phenyl ether to 2-allyl alcohol proceeds through a are distinguished cyclohexadienone intermediate, which then undergoes a keto- in this figure by enol tautomerization. blue and green colors. 81 82

Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Use TSA to understand the chair mechanism The structure of E. coli chorismate mutase of chorismate mutase • Chorismate mutase is a homodimer. Jeremy Knowles has shown that both the chorismate mutase and its • The active site is formed by each dimer! uncatltalyze d so ltilution coun terpar t proceed via a chair mechanism.

83 84 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture The Chorismate Mutase Active Site Favors a NAC • Figure 14.33 Chorismate bound to • TSA stabilization by the active site of – Twelve electrostatic and chorismate mutase in a hydrogen-bonding structure that interactions resembles a NAC. – Arrows: hydrophobic interactions – Red dotted lines: electrostatic interactions.

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Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture Formation of a NAC is facile in the To see is to believe! chorismate mutase active site

Figure 14.34 • A High-Energy Intermediate in the Phosphoglucomutase Chorismate mutase Reaction was seen by X-ray diffraction! fac ilita tes NAC • Transition state formation. The has very short energy required to move from the NAC lifetime? Why we to the transition state could see it? is essentially equivalent in the catltalyzed and uncatalyzed reactions.

87 88 Hanjia’s Biochemistry Lecture Hanjia’s Biochemistry Lecture EndofPart3End of Part 3 End of this class

• Ask yourself… • You should know… – What is the catalytic mechanism of serine – Whyyy enzyme could accelerate a reaction? protease? • 2 major reasons – What is the catalytic mechanism of aspartic – How enzyme could accelerate a reaction? protease? • 5 mechanisms – What is the catalytic mechanism of – Examples of enzyme catalysis mechanism chorismate mutase? • Remember serine protease!

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