Biochemistry for Environmental Health

UNIVERSITI TEKNOLOGI MARA FACULTY OF HEALTH SCIENCES

BIOCHEMISTRY FOR ENVIRONMENTAL HEALTH

INSTITUT PERKEMBANGAN PENDIDIKAN (InED) UNIVERSITI TEKNOLOGI MARA (UiTM) 40450 SHAH ALAM STUDY GUIDE for BIOCHEMISTRY 2009

BIOCHEMISTRY FOR ENVIRONMENTAL HEALTH (ENV 400/416)

Bachelor in Environmental Safety and Health (Honours) Program (e- pjj) Faculty of Health Sciences Universiti Teknologi Mara (UiTM)

2 STUDY GUIDE for BIOCHEMISTRY 2009

Course Description:

This is an introduction to the chemistry of biological compounds. A systematic study of carbohydrates, lipids, amino-acids, proteins, nucleic acids, and their components is presented. Metabolism of the biological compounds is also studied as are the interrelations among the carbon, nitrogen, and energy cycles. Enzymology, intermediary metabolism, and metabolic control will also be included.

Course Outcomes:

Upon successful completion of this course the student should be able to:

1. Explain the different types of bonding and interactions found in biochemistry such as hydrogen bonds, ionic bonds, hydrophobic interaction, Van der Waals forces and asymmetry of carbon compounds with cis-trans isomerism. 2. Explain that water molecules are polar and form irregular hydrogen-bonded networks in liquid state and why polar and ionic substances dissolve in water. 3. Explain how acids and bases affect the pH of a solution, the relationship between pH and pK for a solution of weak acid and how the buffer works.

4. Describe the structure of an amino acid and the structure of the 20 different R groups. 5. Describe the structure of alpha helix, beta sheet primary, secondary, tertiary and quartenary and the covalent and non-covalent forces that maintain structures.

6. Describe the metabolic disorder, phenyketonuria. 7. Explain how the Michaelis-Menten equation relates the initial velocity of a reaction for an enzyme substrate reaction and Lineweaver plot can be used to present kinetic data. 8. Describe competitive and non-competitive inhibitor. 9. Explain the common features in amino acid biosynthesis and the role of urea cycle in amino acid breakdown.

3 STUDY GUIDE for BIOCHEMISTRY 2009 10. Explain the levels of nucleic acid structure and the structure and functions of DNA and RNA

11.. Describe how monosaccharide cyclize to from two different anomers and the monosaccharide linkages in polysaccharides. 12. Describe lactose intolerance, diabetes and hypoglycemia 13. Describe glycolysis and the citric acid cycle to synthesize ATP and some allied health perspective of anerobic metabolism with dental plaque. 14. Describe electron carriers and how electrons travel from the different complexes. 15. Describe the connection between Electron Transport Chain and Oxidative Phosphorylation.

16. Describe the structure and nomenclature of lipids including fatty acids, triacyglycerols, sphingolipids and phophoglycerides.

17. Explain the physiological roles of lipids as membrane components and energy storage molecules.

18. Explain lipid of lung surfactant.

19. Explain fatty acid synthesis and degradation.

20. Explain cholesterol biosynthesis and atherosclerosis

CONTENTS PAGE

1.0 Basic Aspects of the chemistry of life 1.1 Biochemistry as the chemistry of living systems 1.2 Asymmetry of carbon compounds and cis-trans isomerism 1.3 Different types of bonding such as hydrogen bonds, ionic bonds hydrophobic interactions, Van der Waals forces

2.0 Water, acid and base, buffer 2.1 Physical properties of water 2.2 Biological importance of water as a solvent 2.3 Hydrogen ion concentration and pH of biological systems 2.4 Relationship between pH and pK for a solution of weak acid 2.5 Physiological buffer systems

3.0 Amino acids and Proteins 3.1 Overall structure and properties of the 20 different R groups. 3.2 Ionizable groups in amino acids. 3.3 Peptide bonds link amino acid residues in a polypeptide 3.4 The structure of primary, secondary, tertiary and quartenary proteins and the covalent and non-covalent forces that maintain structures. 3.5 The metabolic disorder, phenyketonuria

4.0 Properties of Enzymes 4.1 Classification and general catalytic properties of enzymes. 4.2 Michaelis-Menten equation relates to the initial velocity of a reaction for an enzyme substrate reaction 4.3 Lineweaver plot to present kinetic data. 4.4 Competitive and non-competitive inhibitor and examples.

5.0 Nitrogen metabolism 5.1 Common features in amino acid metabolism 5.2 Glucogenic and Ketogenic amino acids 5.3 The role of urea cycle in amino acid breakdown

6.0 Sugar and carbohydrate structure 6.1 Monosaccharide and their derivates. 6.2 Cyclization to from two different anomers and glycosidic bond that links two monossacrides. 6.3 Dissacharides and other sugars example as sweeteners 6.4 Polisaccharides such as starches and glycogen, cellulose. 6.5 Lactose intolerance, diabetes , hyphoglyceamia and hyperglycemia.

7.0 Metabolic processes central to ATP synthesis- Glycolysis and Citric acid cycle 7.1 Glycolysis involves the breakdown of glucose to pyruvate to synthesize ATP 7.2 Aerobic and anaerobic metabolism. 7.3 The citric acid cycle, a multistep catalytic process that converts acetyl groups to NADH, FADH and GTP. 7.4 Allied health perspective of anaerobic metabolism

8.0 Electron transport and oxidative phosphorylation 8.1 Electron carriers as electrons travel from the different complexes. 8.2 The reactions catalyzed by the complexes and their mechanism 8.3 The connection between electron transport chain and oxidative phosphorylation.

9.0 Lipid and Membranes 9.1 Structure and nomenclature of lipids including fatty acids, triacyglycerols, sphingolipids and phophoglycerides 9.2 the physiological roles of lipids as membrane components and energy storage molecules. 9.3 Lipid of lung surfactant

10.0 Lipid metabolism 10.1 Steps of fatty acid synthesis and its mode. 10.2 HMG-CoA is important in cholesterol biosynthesis. 10.3 Atherosclerosis

11.0 Nucleotides, nucleic acids 11.1 Levels of nucleic acid structure – nitrogenous bases, nucleosides, nucleotides. 11.2 Structure and functions of DNA and RNA . 11.3 Use of nucleoside analogues as drugs

SYLLABUS CONTENTS

CHAPTER 1 : Basic aspects of the chemistry of life

1.1 biochemistry is the chemistry of living systems: 1. complicated and highly organized 2. each part has a function 3. function is related to structure 4. must extract energy from the environment

chemicals of living systems • alcohols

• esters

• ethers

• amides

• acids

• anhydrides

• also include thiols and phosphates

Biochemistry deals with the structure and function of biomolecules biochemists study the structures of bio-molecules and their cellular functions to better understand living systems and their chemistry Example of structure-function relationship 1. amino acids are joined to form proteins and these proteins fold up to form functional enzymes 8 STUDY GUIDE for BIOCHEMISTRY 2009 2. nucleotides are joined to form Rna and Dna. these polymers are the information molecules of living systems and maintain the genetic heritage of organisms 3. proteins (enzymes), RNA and DNA along with other molecules aggregate to form cellular components, cells, organs and whole organisms.

• Rna comes in 3 basic forms:

• tRNA (transfer rna) = adapter in protein synthesis - matches codon to amino acid • Rrna (ribosomal RNA) = structural RNA in ribosomes

• mRNA (messenger rna) = contains information for protein synthesis

cell structure basics of the relationship between proteins and DNA:

• linear relationship between DNA, RNA and protein sequence

• DNA encodes amino acids of a protein using 3 letter codons.

• DNA is transcribed to make mRNA. mRNA is translated by ribosomes to make the protein.

Biochemistry can be divided into 3 areas of study • conformational- structure and 3d arrangements of biomolecules

• metabolism – energy production and utilization

• informational- language for communication inside and between cells

Practical applications of biochemistry

1. in medicine and health care : • enzymes as markers for disease eg lactate dehydrogenase (heart attack can diagnosed by an increase of ldh from heart muscle • acetylcholinesterase (ace) important in controlling certain nerve impulse. many pesticides interfere with this enzyme. • designer drugs – new and improved antibiotics and chemotherapy agents

• human proteins through genetic recombinant techniques eg insulin, hgh 2. in agriculture – herbicides & pesticides, genetic engineering 3. chemical industries – synthesis & detoxification

Biochemical connections • lactic acid and sports

• neurophysiology – some aa are key precursors to hormones and neurotransmitters • nutrition- aspartame (sweetener), lactose intolerance

• allied health- phenylketonuria, multiple sclerosis, lupus (autoimmune disease /immune system attacks the body own tissues involve rna processing), dental plaque,anemia, atherosclerosis • forensic- uses of DNA testing

CHAPTER 2 : Water, acid and base, buffer

Water • essential for life

• major constituent of almost all life forms

• most animals and plants contains more than 60% water by volume

• structure consists of 2H atoms bonded to 1 O atom.

• the H side of the molecule has a slight +ve and the O side a –ve charge exist • makes it polar and has strong solvent properties

• hydrophilic compounds interact (disslove) with water eg . polar cpds (alcohols and ketones)& ionic cpds (kcl), amino acids • hydrophobic compounds do not interact with water eg. non polar cpds (hexane, fatty acids, cholesterol)

Roles of water in the life of organisms

• mammalian cells 70% water

• solvent for biological systems & for most chemical reactions that support life. • 75% of the earth is covered with water

• has a very high specific heat-retains heat better than other materials Some uses of water as solvent • flavoring and co2 gas dissolved in water to make soft drinks

• farmers use water to dissolve fertilizers

• medicines in water

12 STUDY GUIDE for BIOCHEMISTRY 2009 • chlorines or flourides added to water

Hydrogen bonds

• water molecules are hydrogen bonded

• the ability to form strong h bond is responsible for the many unique characteristics of water such as its high melting point and boilng point • 3d structures of many important biomolecules including proteins (Hb) and nucleic acids (DNA) are stabilized by H bonds

Acids, bases, and buffers Principle of ionization of weak acids: • the fundamental concept of buffers is: a buffer resists change

• pH buffers resist change in ph when either acid (h+) or base (oh-) is added to it. • chemicals which are ph buffers are weak acids or bases

• acids = proton (H+) donors

• bases = proton acceptors

This tendency to ionize can be put in terms of an equation for the equilibrium:

where [ ] = molar concentration; k = ionization constant (acid dissociation constant)

Simplest example is water (H2O):

but since [H2O] (water concentration) = constant (55.5 m), kw = [h+][oh-] = 10-14 M in pure water, [h+] = [oh-] = 10-7 m • to make this easier to use, the ph scale was invented.

• pH = -log [h+]; thus when [h+] = 10-7 m, ph = 7

• this is called neutral ph because it is in the middle of the ph scale. at ph greater than neutral, the solution is alkaline; while at ph less than neutral, the solution is acid.

14 STUDY GUIDE for BIOCHEMISTRY 2009 TITRATION OF A WEAK ACID ILLUSTRATING ITS IONIZATION AND BUFFERING PROPERTY

• all weak acids have titration curves like this one. bases (like ammonium, nh4+) are also weak acids and have similar titration curves. • the position where the buffering zone is on the ph scale is related to the chemical nature of the weak acid: • acetic acid ionizes in the acidic portion of the pH scale

• this relationship is known as the Henderson-Hasselbalch equation.

• useful in predicting the properties of buffer solutions used to control the pH of reaction mixtures. • the pk of a weak acid is the ph where [a-] = [ha]

• at pH below the pk, [HA] > [A-]

• at pH above the pk, [HA] < [A-]

• therefore the pk determines the buffering zone for a weak acid.

• a similar expression pk can be used, pk=-log k

• the ph of a solution of a weak acid and its conjugate base is related to the concentration of the acid and base- Henderson- Hasselbach equation.

16 STUDY GUIDE for BIOCHEMISTRY 2009 • for example, acetic acid has a pk = 4.8 and a buffering zone from ph 3.8 to 5.8. • so a weak acid will be an effective buffer at ph = pk +/- 1 ph unit. summary • acids are proton donors and base are proton acceptors

• water can accept or donate protons

• the strength of an acid is measured by its acid dissociation constant, k

• the larger the k value, the stronger the acid and more h+ dissociates

• the conc. h+ is expressed as ph, -ve log of H ion conc.

Calculating pH for weak acids and bases Calculate the relative amounts of acetic acid and acetate ion present at the following points when 1 mol of acetic acid is titrated with NaoH. use HH eqn. to calculate ph 1. 0.1 mol NaOH added 2. 0.3 mol NaOH added 3. 0.5 mol NaOH added ratio 1:1, when 0. 1 mol of naoh added, 0.1 mol acetic acid reacts with it to form 0.1 mol acetate ion, leaving 0.9 mol acetic acid ph = pk + log 0.1/0.9 = 4.76 -0.95 = 3.81

TUTORIAL 1, ENV 416/400

1. Calculate the hydrogen ion concentration for each of the following materials:

a) Blood plasma, pH 7.4 b) Orange juice, pH 3.5 c) Human urine, pH 6.2 d) Household ammonia, ph 11.5 e) Gastric juice, pH 1.8

2. Define the following : a) Acid dissociation constant b) Equivalence point c) Hydrophilic d) Hydrophobic e) Non polar f) Polar

- 3. What is the [CH3COO ] / CH3COOH ratio in an acetate buffer at pH 5.00?

4. What are some macromolecules that have hydrogen bonds as part of their structures?

5. What is the relationship between pKa and the useful range of a buffer?

6 What is the pK of a weak acid HA if a solution containing 0.2M HA and 0.1M A- has a pH 0f 6.5 ?

7. Explain buffer solution. Give an example of a buffer solution.

8. Explain why polar substances dissolve in water while non polar substances do not.

9. Explain why a 1M solution of HCl has a pH of 0.

10. A 5.0 ml of H2SO4 is titrated with 0.2 M KOH to neutrality. If 4.5 ml of KOH was used what was the pH of the original acid?

CHAPTER 3 : Amino acids and Proteins

Amino acids and peptides

1. Only 20 aa usually found in proteins

2. The general structure includes an amino group and a carboxyl gp.

3. The α-carbon is bonded to a H and side chain gp (R)

4. The R gp determines the identity of the particular amino acid

Types of Amino Acids based on side-chain chemical character: I. Non-Polar or hydrophobic (water hating) II. Flexible III. Polar or hydrophilic (water loving)

There are 20 Amino Acids encoded by codons in the genetic code:

When there are more than 100 AAs found in nature, why only 20 AAs in proteins? Because these 20 AAs provide all the chemical and size groups needed to make a very large number of proteins. Plus many of these amino acids become modified after translation into proteins, which increases the available chemical character of amino acid side chains.

These 20 AAs can be divided into the above 3 groups (non-polar, flexible and polar) and then subdivided by their chemical character:

Group I = Non-Polar -- 8 AAs 19 STUDY GUIDE for BIOCHEMISTRY 2009

Hydrocarbon NON-POLAR AMINO ACIDS -- 5 AAs -- Ala Val Leu Ile Pro: Non-Polar -- Hydrocarbon -- Ala (Alanine)

The chiral Carbon of Ala is emphasized here! All amino acids are derivatives of Ala, except Gly

Non-Polar -- Hydrocarbon -- Val (Valine)

Val has to methyl groups added to Ala to make an isopropyl group.

Non-Polar -- Hydrocarbon -- Leu (Leucine)

Leu adds an isopropyl group to Ala so that Leu has 4 carbons in its side chain. Non-Polar -- Hydrocarbon -- Ile (Isoleucine)

Ile is a structural isomer of Leu so it also has 4 carbons in its side chain. But Ile is bulkier than Leu near the base of the side chain, while Leu is bulkier than Ile 20 STUDY GUIDE for BIOCHEMISTRY 2009 farther out on the side chain (size/shape of side chains is important). Ile has a 2nd chiral center which is emphasized in the Ile drawing above.

Non-Polar -- Hydrocarbon -- Pro (Proline)

Pro is a very special amino acid due to its inflexible character!!! Pro is inflexible because its side chain bonds to alpha-amino group in a ring structure which can not twist around the bond between alpha-amino group and alpha carbon, which all other AAs can. Also Pro, thus, has a secondary amino group (notice the single hydrogen on its Nitrogen atom) with different chemical character than the primary amino groups in all other amino acids, which have two hydrogens on them.

Aromatic NON-POLAR AMINO ACIDS -- 2 AAs --Phe Trp: Non-Polar -- Aromatic -- Phe (Phenylalanine)

Phe adds a benzene ring to Ala!

Non-Polar -- Aromatic -- Trp (Tryptophan)

Trp has a heterocyclic aromatic group with an aromatic amine in it.

Thiol Ether NON-POLAR AMINO ACID -- 1 AA -- Met: Non-Polar -- Thiol Ether -- Met (Methionine)

Met introduces the important Sulfur element into proteins which is found in Cys also (see below). Met contains a thiol ether (R-S-R) in its side chain, which is much less polar than an oxy-ether (R-O-R) like the compound we call ether, which is an polar organic solvent. Met is a very hydrophobic AA.

Group II = Flexible -- 1 AA -- Glycine is the Flexible Amino Acid Flexible -- Gly (Glycine)

22 STUDY GUIDE for BIOCHEMISTRY 2009 Gly is a unique AA with no chiral center -- but it is prochiral since it has two groups the same (ie H) on the central carbon -- so it still has sidedness - try making a model of Gly. Most important since Gly has no side chain it is very flexible and can easily twist around its alpha-amino Nitrogen bond to the alpha- Carbon. Gly is the opposite of Pro - Gly is flexible while Pro is inflexible. Finally, Gly makes a transition from the non-polar AAs to the polar AAs. Gly is neither nonpolar or polar .

Group III = Polar -- 11 AAs THE POLAR AMINO ACIDS Polar AAs are important since they provide chemical groups for interaction with water. Thus, the hydrogen bonding character of polar AAs is key in forming protein structures. While the ionic bonding character of charged polar AAs is also important in protein structure. Also the polar side chains in these AAs provide the chemically reactive groups in proteins.

Alcohols - Neutral Polar Amino Acids -- 3 AAs -- Ser Thr & Tyr: Polar -- Neutral -- Alcohols -- Ser (Serine)

Ser contains one -OH group and so it is essentially hydroxy-Ala. The hydroxyl group on Ser does not normally ionize, so it is not charged in proteins - its neutral. Ser is the smallest AA of the polar amino acids and is very polar. The hydroxyl group on Ser provides enzymes a very good nucleophilic group for doing chemistry. Another important function of Ser is to form esters with phosphate, making phospho-ester proteins. Phosphorylation of proteins/enzymes is very important in regulation of activity.

Polar -- Neutral -- Alcohols -- Thr (Threonine)

Thr adds a Carbon on to Ser, which makes the hydroxyl group less accessible in Thr than Ser. Thr serves more often in a structural role in proteins and is usually not as chemically active as Ser. Thr can form esters with phosphoric acid and phospho-Thr is often found in proteins. 23 STUDY GUIDE for BIOCHEMISTRY 2009

Polar -- Neutral -- Alcohols -- Tyr (Tyrosine)

Tyr is an aromatic alcohol and so it has both aromatic character and polar character. The hydroxyl of Tyr is like the hydroxyl in phenol, so at high pH it can ionize. Tyr can also form phospho-esters like Ser and Thr. Phospho-Tyr is very important in proteins/enzymes involved in regulating the cycle cell.

Thiol - Neutral Polar Amino Acid -- 1 AA -- Cys: Polar -- Neutral -- Thiol -- Cys (Cysteine)

Cys is essentially thiol-Ala. The thiol (-SH) group of Cys can ionize as shown in graphic. Thiols ionize at about pH 8 and so usually they are protonated at biological pH. Hydroxyl groups like in Ser have pK about 15 or so and do not ionize normally. A Special Feature of Cys is that it can oxidize (in the presence of oxygen) and 24 STUDY GUIDE for BIOCHEMISTRY 2009 react with another Cys to form Cystine or a disulfide bond:

The formation of "Cystine" can take place between 2 polypeptide chains to make a cross-link between them. This is actually an enzyme catalyzed reaction which takes place in the lumen of ER in cells when proteins are being exported from the cell. A very good example is the production of antibodies by cells in the immune response - antibody proteins contain many Cys- Cys or disulfide bonds. Excellular proteins often contain Cys-Cys bonds, while cellular proteins do not usually contain the Cys-Cys since the conditions in the cell are reducing. In the second part of the graphic above, the general reaction of 2 thiols is shown. In the presence of oxygen or oxidizing conditions, the 2 thiols react to form a disulfide bond between them. Since this is a redox reaction, the hydride ion released by each thiol is usually coupled to an electron acceptor reaction or in simple oxidiation with oxygen, hydrogen peroxide is usually formed with further reduction to water.

Amides - Neutral Polar Amino Acids -- 2 AAs -- Asn & Gln: Polar -- Neutral -- Amides -- Asn (Asparagine)

Asn is a very small amino acid as well as being very polar. Amides are neutral and do not ionize nor do they accept protons.

Polar -- Neutral -- Amides -- Gln (Glutamine)

Gln is a bit larger amide than Asn because it has a longer side chain string of Carbons. Both the amide AAs are neutral derivatives of the corresponding acid AAs (Asp & Glu - see below) Understanding the chemical character of the amide is very important, since the peptide bond of proteins is an amide bond.

Acids - Negatively Charged Amino Acids -- 2 AAs -- Asp & Glu: Polar -- Charged -- Acids -- Asp (Aspartic acid or Aspartate)

Asp has a second carboxylic acid group in addition to its alpha-carboxylic acid group. The Asp side chain carboxyl group is normally ionized at biological pH; Asp a negatively charged AA. Asp is a rather small AA and is very polar.

Polar -- Charged -- Acids -- Glu (Glutamic acid or glutamate)

Glu also has a second carboxylic acid group in addition to its alpha-carboxylic acid group. The Glu side chain carboxyl group is normally ionized at biological pH; Glu is negatively charged.

Bases - Positively Charged Amino Acids -- 3 AAs -- Lys Arg & His: Polar -- Charged -- Bases -- Lys (Lysine)

Lys has a primary amino group at the end of a 4 Carbon side chain and it can be positively charged. Since the Lys side chain amino group has a high pK , it is often charged at biological pH.

Polar -- Charged -- Bases -- Arg (Arginine)

Arg has a complex side chain containing 3 Nitrogen groups, which work as a unit to give a positive charge. Since the Arg side chain group has a very high pK , it is always charged at biological pH. Arg provides proteins/enzymes with essentially a fixed positive charge.

Polar -- Charged -- Bases -- His (Histidine)

His has an aromatic-like pair of amino groups, making His a unique AA with a positive charge -- sometimes. His with a pK for its side chain near neutrality, means that it can either be charged or not at biological pH. His, when not charged, is a very strong nucleophile and is very important in enzyme chemistry. His is also very important as a proton acceptor and donor in biochemical reactions.

Protein Covalent Structure (Protein Primary Structure) I. Peptide Bonds, Peptides and Proteins Proteins are sometimes called Polypeptides, since they contain many Peptide Bonds The peptide bond is an amide bond Water is lost in forming an amide bond. Structural Character of Amide Groups: Understanding the chemical character of the amide is very important, since the peptide bond of proteins is an amide bond. Amides have a partial double bond character and also a partial charge character because of the resonance forms shown in the above graphic. Comparison of an amino acid, a dipeptide and a tripeptide: Amino Acid = Gly; dipeptide = Gly-Ala; tripeptide = Gly-Ala-Ser Peptides = Mini-Proteins A pentapeptide -- GlyAlaSerPheGln 1st amino acid is always written on the left and called the Amino terminal, since it is always the only amino acid of the peptide with a free alpha-amino group. Last amino acid is always written on the right and called the Carboxyl terminus, since it is always the only amino acid of the peptide with a free alpha-carboxylic acid group.

List of Proteins Shown in Amino Acid Composition Table: A. Antibody - Human Bence-Jones Kappa (antibody light chain) B. Human Cytochrome c (electron transport protein) C. Spinach Ferredoxin (electron transport protein) D. Pig Glucagon (protein hormone) E. Bovine Insulin (protein hormone) F. Human/Gorilla Hemoglobin alpha chain (oxygen transport protein) G. Human/Gorilla Hemoglobin beta chain (oxygen transport protein) H. Chicken Lysozyme (enzyme) I. Sheep Wool (structural protein) Free amino acids are obtained from proteins by strong acid hydrolysis:

B. OVERALL CONFORMATION OF PROTEINS

Proteins have a covalently bonded backbone as discussed in Lecture 5 in relation to amino acid sequence determination. But the 3-D shape or conformation is held together by weaker bonding of the non-covalent type. The linear form of the polypeptide backbone of the protein folds into a tightly held shape which is chemically stabilized by weak bonds like hydrogen bonds, ionic bonds and hydrophobic interactions among non-polar amino acid side chains. To reduce the complexity of protein structure to a manageable level for our study and understanding, the protein is considered to have 4 levels of structure.

Four Levels of Protein Structure: 1. Primary Structure- Polypeptide backbone 2. Secondary Structure- Local Hydrogen bonds along the backbone 3. Tertiary structure- Long distance bonding involving the AA side chains 4. Quaternary structure- Protein-Protein interactions leading to formation of dimers, tetramers, etc.

C. PRIMARY STRUCTURE OF PROTEINS We have already discussed the Primary structure of Proteins, which is the polypeptide backbone or amino acid sequence. The amide bonds joining the individual amino acid residues of the backbone have an important role in forming the 3-D structure of proteins. The peptide bond of the amino acid sequence forms a planar structure due to the partial double bond between N and C. This planar structure limits the ways the backbone can fold up and therefore, constrains the shape a folded polypeptide can take.

29 STUDY GUIDE for BIOCHEMISTRY 2009 The Amide Bond showing its partial double bond character and partial charges.

D. SECONDARY STRUCTURE OF PROTEINS

In 1950's, Linus Pauling named the first structures he found by X-ray diffraction, the Alpha Helix and the second structure he found was called Beta Sheet. We continue to use these names today for two forms of secondary structure and add a third type forms in regions where the protein bends back on itself to form its compact shape or conformation. The 3 Types of Protein Secondary Structure: Alpha-helix Beta-sheet Turns or Bends (Bends in backbone to fold the polypeptide back on itself)

E. LOCAL HYDROGEN BONDING FORMS SECONDARY STRUCTURE

Secondary Structure is formed by local Hydrogen Bonding between the Hydrogen on the Nitrogen of one amide in a peptide bond with carbonyl oxygen of another amide in a second peptide bond. Hydrogen bonds (H-bonds)are weak non-covalent bonds. The energy required to break an Hbond is about 1 to 4 kcal/mole as compared to a covalent bond which requires about 100 kcal/mole to break. Thus, H-bonds are a bit flexible and for example, the H-bonds holding water together as liquid constantly break and reform. However, in more directed H-bonds like found in protein secondary structure, the pair of groups involved stay as partners and with the overall arrangement of a single H-bond being stabilized by a group of H-bonds. So H-bonding in secondary structure is stronger due to the local grouping of these bonds and secondary structure forms like the alpha-helix and beta-sheet are neighborhoods of H-bonds acting together like a group.

Figure 7. Hydrogen Bond (H-Bond) between Two Peptide Bonds.

Model of Right-Handed Alpha-Helix Showing H-Bonding (From Voet/Biochemistry 1990 John Wiley)

F. Alpha-HELIX

Alpha helix is held together by hydrogen bonds between the amide Hydrogen on the Nitrogen and another amide carbonyl oxygen of every 4th amino acid residue (approximately). These are intrachain H-bonds which along the same region of the backbone of the polypeptide or in other words within the same region of the amino acid sequence.

Simple Model of Alpha Helix with H-bonding Pattern.

The side chains of the amino acids project out from the core of the alpha helix. Water is excluded from the tight inner core of the alpha helix, which is very hydrophobic.

G. Beta SHEET Beta sheets are also held together by hydrogen bonds between the Hydrogen on the Nitrogen and another amide carbonyl oxygen of the peptide bonds but between chains of the backbone rather than along it as was found for the Alpha helix. These are called interchain H-bonds since they form between two parts of the polypeptide backbone separated from one another by some distance or length of the amino acid sequence of the polypeptide.

Simple model of H-Bonding in a Beta Sheet.

Two types of backbone chain order is found: 1. PARALLEL where the chains run in the same direction 2. ANTI-PARALLEL where chains run in the opposite direction

34 STUDY GUIDE for BIOCHEMISTRY 2009 H. TURNS AND BENDS IN THE POLYPEPTIDE BACKBONE

Proline (Pro) breaks up secondary structures like alpha-helix and beta-sheet. Because Pro can not bend, Pro is often found at the ends of Alpha Helix and Beta Sheet strands. Thus, the third type of Secondary Structure is actually formed by the absence of the other two types.

Positions of Pro in Relation to Alpha-Helix and Beta Sheet Secondary Structures

Places where the polypeptide backbone bends so that the protein can fold back on itself to form the compact structure also have hydrogen bonds in some cases. These H-bonds occur only between the 1st and 4th amino acid residue of the Reverse Turn and no other H-bonds are formed.

TUTORIAL 2, ENV 416

1. Draw the dipeptide Asp-His

2. Identify the nonpolar amino acids and the acidic amino acids in the following peptide :

Glu-Thr-Val-Asp-Ile-Ser-Ala

3. Sketch a titration curve for alanine and indicate the pKa values for all the titratable groups. Also indicate the pH at which this amino acid has no net charge.

4. Draw 2 hydrogen bonds, one is part of a secondary structure and another that is part of a tertiary structure. 35 STUDY GUIDE for BIOCHEMISTRY 2009

5. Draw a disulfide bridge between two cysteines in a polypeptide chain.

6. What is the highest level of oragnization in myoglobin and hemoglobin?

7. Differentiate between secondary and tertiary proteins. Name an example for each.

8. Differentiate between alpha-helix and beta sheet.

CHAPTER 4 : PROPERTIES OF ENZYMES

Enzymes are biological catalysts. Like all catalysts, enzymes lower the energy needed to get a reaction started. Enzymes are much generally better at accelerating the rates of reactions than non-biological catalysts.

Figure 1. Diagram showing that less energy is required to get an enzyme catalyzed reaction started as compared to a non-catalyzed reaction. Figure from Zubay et al., Principles of Biochemsitry copyright 1995 Brown Comm.

Enzymes have been divided into 6 classes by the International Commission on Enzyme Nomenclature. All enzymes are assigned a number (called an EC number) which defines exactly the reaction catalyzed by the enzyme. For example, trypsin is EC 3.4.21.4 since it is in class 3 (hydrolases) which work on peptide bonds (3.4) in the middle of proteins (3.4.21 are serine endopeptidases) - trypsin is the 4th entry in this subclass.

These six classes are: 1. Oxidoreductases - enzymes catalyzing oxidation reduction reactions. 2. Transferases - enzymes catalyzing transfer of functional groups. 3. Hydrolases - enzymes catalyzing hydrolysis reactions. 4. Lyases - enzymes catalyzing group elimination reactions to form double bonds. 5. Isomerases - enzymes catalyzing isomerizations (bond rearrangements). 6. Ligases - enzymes catalyzing bond formation reactions couples with ATP hydrolysis. These 6 enzyme classes can also be illustrated by the general reactions catalyzed:

Examples of enzymes in each class: 1. Alcohol dehydrogenase (EC 1.1.1.1) 2. Hexokinase (EC 2.7.1.1) 3. Trypsin (EC 3.4.21.4) 4. Ribulose-bisphosphate carboxylase (EC 4.1.1.39) 5. Triose phosphate isomerase (EC 5.3.1.1) 6. Tyrosine tRNA ligase (6.1.1.1)

Enzyme Additives (Cofactors) Assisting in Catalysis Enzymes are often composed of only protein. In this case only AA side chains are used for catalysis. Some enzymes require additives for assisting with catalysis. Additives like vitamins often provide functional groups not available to the enzyme among the side chains of the amino acids. In these cases the protein of the enzyme binds: Organic cofactors (Vitamins = organic cofactors) Metal ions (e.g. Mg2+) Nucleotides (even RNA) The Common Cofactors (Enzyme Additives): Biotin aids in carboxylation reactions (carbon dioxide fixation).   Cobaltamine (vitamin B-12) aids in alkylation reactions (methylation for instance). Coenzyme A aids in acyl transfers like in the tricarboxylic acid cycle. Flavin (vitamin B-2) aids in oxidation-reduction reactions (e.g. nitrate reductase). Lipoic acid aids in acyl transfers via oxidation-reduction processes. Nicotinamide coenzymes like NAD+ act as independent co-substrates.   Pyridoxal (vitamin B-6) aids in amino group transfers (provides aldehyde functional group). Tetrahydrofolate aids in one-carbon transfers.   Thiamin (vitamin B-1) aids in aldehyde transfers and alpha-keto-acids decarboxylations

The complex of protein and additive is called Holo-Enzyme. When the additive is removed from the enzyme, the remaining protein part of the enzyme is called the Apo-Enzyme. Apo-Enzyme (inactive) + Additive = Holo-Enzyme (active)

The Active Site of the Enzyme. Each enzyme has a unique active site. Active site = catalytic site.

38 STUDY GUIDE for BIOCHEMISTRY 2009 The enzyme binds its substrate(s) at the active site and the enzyme catalyzes chemical changes in the substrate(s). The types of chemical reactions catalyzed were illustrated above in

Figure 3. NAD+ bound in the active site of GAP dehydrogenase. The NAD+ molecule is shown in bold and the side chains of the amino acids binding it are shown projecting from the surface of the enzyme (shown as the filled in area surrounding the active site).

Enzymes contain a large number of amino acids, but most AA side chains are used for forming the enzyme's shape. Only a few AA side chains are at the active site. These special AA side chains: 1. Bind the substrate(s) and 2. Catalyze the reaction This concept is illustrated in the following figures by 3 different drawings of the enzyme

39 STUDY GUIDE for BIOCHEMISTRY 2009 ribonuclease which catalyzes the hydrolysis of RNA. The first view is of the 3-D shape of the enzyme with the 3 key amino acids at the active site highlighted (His12, Lys41 and His119 - numbers indicating the position of these residues in the amino acid sequence of ribonuclease). Next is a ribbon model with the 3 key amino acids shown in relation to the various secondary structure elements of ribonuclease. Last is a ball-and-stick model of ribonuclease with the same 3 amino acid side chains of the active site emphasized. A feature to try to see in these models is the groove of the enzyme which forms the active site and how the enzyme folds to bring these 3 key amino acid side chains together to form the active site. Figure 5. 3-D model of the enzyme ribonuclease with the key amino acid side chains at the active site shown in red. The active site is a deep groove at the center of this structure.

Summary of the Active Site of Enzymes: Enzyme has large structure with hundreds of AA side chains but only a few are involved in catalysis. Each enzyme has a unique active site. Key AA side chains are involved in binding and catalysis in the active site.

Enzyme Framework - Why are Enzymes so Large? We have discussed the formation of a protein's 3-D shape recall- the 4 levels of protein structure: Primary, Secondary, Tertiary, Quaternary. They make up a "Framework" to bring the AA side chains of the active site together. By bringing the AA side chains of the active site together they can act synergistically or in concert which is part of what makes enzymes very effective catalysts.

Figure 6. Ribonuclease with substrate RNA model bound in active site. His12 and His119 are involved in catalysis of the phosphodiester bond in the backbone of the RNA. Lys41 assists with binding the RNA molecule. The active AA side chains also provide the enzyme with a high degree of specificity so that only certain substrates are bound to the enzyme's active site..

How do enzymes catalyze a reaction??? One answer is: Like all catalysts, enzymes decrease the energy required to get a reaction started. This was illustrated in the first part of this lecture with an energy diagram. Below is shown a similar diagram with more detail for the energy pattern for the enzyme catalyzed reaction. First, energy is required to form the complex between the enzyme and substrate (E-S complex) which is a higher energy state than the free enzyme and substrate/product.

Figure 7. Diagram of energetics of enzyme catalyzed reaction versus non- catalyzed reaction.

Summary of Enzyme Catalysis: Enzymes bind substrate with great specificity Enzyme catalyzed reactions usually have no side products   Enzymes use energy released when substrates bind to make their catalysis more effective.

Introduction to Enzyme Kinetics.

In chemistry, kinetics has to do with the rate of reactions. In biochemistry, we are most interested in rates of enzyme catalyzed reactions since virtually all biological reactions are catalyzed by enzymes.

Enzyme Kinetics: Rates of enzyme catalyzed reactions Usefulness of enzyme kinetics: Common clinical assays to detect enzymes Understanding metabolic pathways Measuring binding of substrates and inhibitors to the active site of an enzyme Understanding the mechanism of catalysis of an enzyme

Rates of reactions are measured by change in reactant amounts with time. You can measure the disappearance of the substrate or the appearance of the product. Usually, the appearance of the product is easier to keep track of since there should be no product present at the beginning of the reaction.

Figure 8. Ways to express a rate for the enzyme catalyzed reaction.

Rates = Reaction Velocity For enzymes, the initial velocity (before significant product accumulates) is always used. Initial Velocity = Vo

A Simple Mechanism for the Enzyme Catalyzed Reaction.

For catalysis to begin, the substrate must bind to the enzyme, which results in the formation of the enzyme-substrate complex (ie E-S complex). The E-S complex forms rapidly in the first part of the enzyme catalysis process and the concentration of the E-S stays constant at a steady-state level. For this reason, this type of kinetics is called steady-state kinetics. A simple mechanism for the enzyme catalyzed reaction helps us to understand and model this process.

E + S ↔ ES → E + P

A simple enzyme mechanism for a single substrate and product.

Enzyme Catalyzed Rates at Different Substrate Concentrations.

Since the enzyme is used many times to catalyze the same reaction, the concentration of the enzyme is much less than the substrate: [S] >> [E] Thus, the substrate saturates the enzyme. This is best understood by observing the rate of the reaction or initial velocity at different [S] (ie. substrate concentrations):

43 STUDY GUIDE for BIOCHEMISTRY 2009 [S] mM Vo μmol product/min 0 0.0 1 0.9 2 1.4 5 1.9 10 2.3 50 2.6 100 2.6

Model data for the enzyme catalyzed reaction. These data show that at low [S], the initial velocity is more or less proportional to the [S]. At high [S], the initial velocity no longer increases as more substrate is added. Thus, at high [S] the enzyme is saturated with substrate and no increase in the enzyme catalyzed rate is observed. This model set of data for an enzyme catalyzed reaction shows the initial velocity in terms of the amount of product formed per unit time (ie micromoles of product produced/min) at various substrate concentrations. These data can be plotted in a graphical form to also illustrate the results of an enzyme catalyzed reaction.

Plot of initial velocity of the enzyme catalyzed reaction (Vo) versus the [S] (ie substrate concentration). Initial velocity is always given in units of amount of product formed per unit time and the substrate concentration is given in molar units (ie mM). Here it is easy to see the saturation of the enzyme at high [S] where the initial velocity approaches a limiting value. The plot has the shape of a square hyperbola.

The Michaelis-Menten Equation.

The plot of Vo versus [S] can be represented by an equation, which is known as the Michaelis- Menten equation in honor of the scientist who first described it. This equation, sometimes called the M-M equation, is an important one for you to know and understand. v0 = Vmax [S ] 44 STUDY GUIDE for BIOCHEMISTRY 2009 Km + [S ]

The Michaelis-Menten equation which describes the change in Vo as [S] increases. The constants in this equation, Km and Vmax, are defined: Vmax = Maximum velocity catalyzed by a fixed [E] Km = the [S] which gives 1/2 Vmax These definitions are illustrated below:

Vo versus [S] plot illustrating the operational definitions of Vmax and Km. Thus, the limit approached in the Vo versus [S] plot is the Vmax.

Definition of Km and Vmax and Their Ratio - Vmax/Km. The Km is sometimes called the Michaelis Constant. The Km is an intrinsic property of an enzyme related to the binding constant for forming the ES complex, which is an equilibrium and can be defined by the rate constants for its formation and breakdown using the simple enzyme mechanism shown above.

The approximate relationship between the Km and the Ks for the binding of the substrate to the enzyme which leads to the formation of the E-S complex. Ks is defined by the equilibrium formed between the enzyme (E) and substrate (S) and the E-S complex, as shown above. Ks is also defined by the ratio of the rate of breakdown of the E-S complex divided by its rate of formation.

45 STUDY GUIDE for BIOCHEMISTRY 2009 But Km also involves the breakdown of the E-S complex to E and P, which is not a component of the Ks. Thus, the rate of the breakdown of the E-S complex to make product (P) is also defined in the simple enzyme mechanism .

The definition of Km by using rate constants for simple enzyme mechanism. The point of this graphic is to emphasize that the Km constant of the enzyme catalyzed reaction includes more than just the formation of the E-S complex, but also its breakdown to form product, which is of course the key to an enzyme catalyzed reaction. So Km reflects both binding of E to S but also the catalytic constant (shown as k3 above, but also defined as kcat) of the enzyme catalyzed reaction.

The Vmax is also dependent on the catalytic constant: Vmax = kcat [E] So both Vmax and Km are properties of individual enzymes and not very useful for comparing enzymes. However, the ratio Vmax/Km can be used to compare enzymes. This ratio (Vmax/Km) measures the efficiency of the enzyme. The efficiency of the enzyme is ultimately limited by the rate of diffusion of the substrate to the enzyme - thus the diffusion of substrates, which is very rapid, sets an upper limit. The most efficient enzymes like Triose-P Isomerase are limited by how fast their substrates get to them. But most enzymes are not this efficient and more limited by chemical events in the active site of the enzyme.

Finding the Km and Vmax by the Graphical Solution Method. To calculate the Km and Vmax, the Michaelis-Menten equation is converted into a linear form by taking the reciprocal of both sides of the equation. This is called the Lineweaver-Burk equation in honor of the first scientists to describe it.

The Lineweaver-Burk equation linearizes the M-M equation by taking the reciprocal of

46 STUDY GUIDE for BIOCHEMISTRY 2009 both sides of the equation. This equation then takes on the form of the equation of a line. The y values are 1/Vo, the x values are 1/[S]. The b value in the line equation is the slope and equal to Km/Vmax, while the c value is the y-intercept and equal to 1/Vmax. The double reciprocal plot is useful for deriving Km and Vmax by plotting kinetic data for an enzyme and you should use it to find the Km and Vmax via graphing for the problem set you got today.

The double reciprocal plot for enzyme kinetic data.

This plot must be used to find Km and Vmax for enzyme kinetic data in this class as shown on the graphic. The y-intercept is the 1/Vmax. The x-intercept, which is found in the 4th quadrant, is -1/Km. Alternatively, the Km value can be found from the slope using the Vmax value found from the y-intercept. However, there are statistical problems with the Lineweaver-Burk equation and double reciprocal plots, so today in research, one derives Km and Vmax using other methods such as the direct linear plot using a computer program. However, the Lineweaver-Burk equation makes the clearest representation of kinetic data and makes it easy to understand the results, so it is most often used to illustrate the data even when the Km and Vmax are derived by other methods.

Enzyme Inhibitors. A. Competitive Inhibition

Inhibitors of enzymes: Two types are considered - Competitive and Non- Competitive. A Competitive Inhibitor has a chemical similarity to the substrate and competes with the substrate for binding to the active site of the enzyme. A good example to describe competitive inhibition is the mitochondrial enzyme, succinate dehydrogenase:

(A) The reaction catalyzed by succinate dehydrogenase is the oxidation of succinate to fumarate. (B) Malonate and oxaloacetate are competitive inhibitors of succinate dehydrogenase.

Both these competitive inhibitors, malonate and oxaloacetate, look like succinate in their chemical character. Both inhibitors are dicarboxylic acids like the substrate succinate so they have groups which can bind in the same places in the active 48 STUDY GUIDE for BIOCHEMISTRY 2009 site of succinate dehydrogenase as the substrate. However, neither inhibitor has the capacity to undergo the reaction and so the enzyme is inhibited. Since these inhibitors simply bind to the enzyme, when the succinate concentration is high, they will be pushed out of the site by the substrate and the enzyme will catalyze the reaction as if no inhibitor were present. An enzyme mechanism model of the action of a competitive inhibitor (Ic) based on the standard model of a Michaelis-Menten enzyme where E + S leads to the E-S complex, which leads to product P:

Model of a Competitive Inhibitor (Ic) Interacting with the Enzyme (E) and an equation for the equilibrium formed between the Ic and E, which is governed by the inhibitor binding constant, Ki.

This model is the same as the one described in the previous lecture where enzyme (E) and substrate (S) bind to form the ES complex, which will go forward during catalysis to form product (P) and the free enzyme. In the presence of the competitive inhibitor, Ic, a complex forms with enzyme when the inhibitor binds, the E-Ic complex. This is a dead-end complex and can not go on to form product. However, the Ic is bound reversibly to the enzyme and when more substrate is added, the inhibition is overcome by pulling the enzyme free via the breakdown of the E-Ic complex, which is in equilibrium with free enzyme and free Ic. Another way to think about this is - when lots of substrate is added, the concentration of free enzyme (E) falls to such a low level, that some of the E-Ic complex must breakdown to replenish the free E demanded by the equilibrium between E and Ic. This can also be demonstrated by comparing the Vo versus [S] plots for uninhibited enzyme and enzyme in the presence of a competitive inhibitor:

Vo versus [S] plot comparing the kinetics of the reaction in the absence of inhibitor and in the presence of the competitive inhibitor (Ic). At high [S], the initial velocity in the presence of Ic will be about the same as it is in the absence of the inhibitor. The concentration of S which will be required to overcome the effect of the competitive inhibitor will depend on the [Ic] (ie. concentration of the competitive inhibitor) and the Ki (ie. the binding constant of the inhibitor to enzyme). In competitive inhibition, addition of more substrate will out compete the inhibitor and overcome the inhibition of the enzyme's catalytic rate - thus, the Vmax will be the same and only Km will be altered. This is most clearly illustrated with the double reciprocal plot comparing the uninhibited reaction to that in the presence of Ic.

Double reciprocal plot for competitive inhibitor (Ic).

Here the uninhibited reaction gives the standard double reciprocal plot from which Km and Vmax can be calculated. The reaction in the presence of the competitive 50 STUDY GUIDE for BIOCHEMISTRY 2009 inhibitor yields apparent constants for the enzyme which are called the Km' and Vmax'. For the true competitive inhibitor, the Vmax' (apparent Vmax for inhibited enzyme) will be the same as the real Vmax, while the Km' (apparent Km for the inhibited enzyme) will be greater than the real Km. Thus, the -1/Km' will be smaller than -1/Km. After finding Km and Km', the Ki for the Ic can be calculated using the equation shown using the given concentration of the competitive inhibitor ([I]).

Enzyme Inhibitors B. Non-competitive Inhibition.

A Non-Competitive Inhibitor does not compete with substrate and the [S] has no influence on the degree of inhibition of the enzyme's catalytic rate. For example, enzymes with a thiol ( -SH ) not at the active site can be inhibited:

Example of a heavy metal inhibiting an enzyme by binding to a thiol group not at the active site and inactivating the enzyme. Non-Competitive Inhibition can be model using the standard model for the Michaelis-Menten enzyme where E + S form the ES complex which leads to formation of product P. In this case where the non- competitive inhibitor (Inc) reacts with the enzyme at a site other than the active site, both the free enzyme (E) and the enzyme-substrate complex (E-S) react with Inc. Clearly, in this case the reaction of the non-competitive inhibitor is irreversible and the substrate can not over come the inhibitors impact on the enzyme:

Model of the Non-Competitive Inhibitor (Inc). The equilibrium between enzyme and Inc now depends on the total concentration of enzyme in all forms present (ie. both the free E and the E-S complex) and defines the Ki. A Vo versus [S] plot for the Non-competitive Inhibitor looks very different than that for a competitive inhibitor since increasing the [S] has no impact:

Vo versus [S] plot for enzyme in the absence and presence of Inc. The double reciprocal plot for this same model shows that Inc decreases Vmax, as if some of the enzyme had been removed from the system. In classic example of pure non-competitive inhibition, the uninhibited reaction and the enzyme in the presence of Inc will yield the same Km value.

Double Reciprocal plot for the Non-Competitive Inhibitor (Inc). Non competitive inhibitors decrease Vmax but have no effect on Km. The apparent Vmax' is smaller than the real Vmax and the Ki for the Non- Competitive Inhibitor can be calculated using the following equation and the known [I]:

Equation showing the relationship between Vmax' (apparent Vmax) and real Vmax in the presence of a Non-Competitive Inhibitor. Use this equation for calculating the Ki of the Non- Competitive Inhibitor at known [Inc].

Evaluating Enzyme Inhibitors to determine type and their Ki. To determine what type an inhibitor is: 1. Find Km and Vmax for uninhibited from 1/Vo vs 1/[S] plot. 2. On same graph find Km' and Vmax' for inhibited reaction. A. If Vmax = Vmax' then inhibitor is competitive type. (Vmax and Vmax' should not be more than 10% different) B. If Vmax does not equal Vmax', then if Km = Km', inhibitor is non competitive type. After finding inhibitor type, then use equations to calculate Ki. Ki is a binding constant for inhibitor to the enzyme. Ki has same units as the [I]. If [I] = mM, then Ki = mM. 53 STUDY GUIDE for BIOCHEMISTRY 2009 Equations used for calculating Ki values:

Equation for Competitive Inhibitor.

Equation for Non-Competitive Inhibitor. Rearrange these equations to solve for Ki.

Tutorial 3, ENV 416

1. An enzyme catalyzed reaction has a Km of 1mM and Vmax of 5 nMs-1, What is the reaction velocity when the substrate concentration is

(a) 0.25 mM

(b) 1.5 mM

2. For an enzymatic reaction, draw a plot to explain how it catalyzes a reaction.

54 STUDY GUIDE for BIOCHEMISTRY 2009 3. (a) Differentiate between competitive inhibitor and non competitive inhibitor .

(b) Which of this is affected by change in the substrate concentration? Why?

4. Calculate Km and Vmax from the following data:

-1 [S] (µM) v0 (mM s ) 0.1 0.34 0.2 0.53 0.4 0.74 0.8 0.91 1.6 1.04

5. Write out the enzyme mechanism model of action for competitive and non competitive inhibitor based on the MM equation.

CHAPTER 5 : Nitrogen metabolism Amino Acid Metabolism

Will be interested in two things: 1) origin of nitrogen atoms and their incorporation into amino group 2) origin of carbon skeletons

AMINO ACID SYNTHESIS 55 STUDY GUIDE for BIOCHEMISTRY 2009

Nitrogen fixation

Gaseous nitrogen is chemically unreactive due to strong triple bond. To reduce nitrogen gas to ammonia takes a strong enzyme --> reaction is called nitrogen fixation. Only a few organisms are capable of fixing nitrogen and assembling amino acids from that. + Higher organisms cannot form NH4 from atmospheric N2. Bacteria and blue-green algae (photosynthetic procaryotes) can because they possess nitrogenase. Enzyme has two subunits: - 1) strong reductase - has Fe-S cluster that supplies e to second subunit 2) two re-dox centers, one of which is a nitrogenase + Composed of iron and molybdenum that reduces N2 to NH4 Reaction is ATP-dependent, but unstable in the presence of oxygen. Enzyme is present in Rhizobium, symbiotic bacterium in roots of legumes (i.e. soybeans) Nodules are pink inside due to presence of leghemoglobin (legume hemoglobin) that binds to oxygen to keep environment around enzyme low in oxygen (nitrogen fixation requires the absence of oxygen) - Plants and microorganisms can obtain NH3 by reducing nitrate (NO3 ) and nitrite - (NO2 ) --> used to make amino acids, nucleotides, phospholipids.

Assimilation of Ammonia

Assimilation into amino acids occurs through glutamate and glutamine. -amino group of most amino acids comes from  -amino group of glutamate by transamination. Glutamine contributes its side-chain nitrogen in other biosynthetic reactions. Reaction:

NADPH +H+ NADP+ + NH4 +  -ketoglutarate glutamate + H2O glutamate dehydrogenase

Another reaction that occurs in some animals is the incorporation of ammonia into glutamine via glutamine synthetase:

+ + glutamate + NH4 + ATP glutamine + ADP + Pi + H

56 STUDY GUIDE for BIOCHEMISTRY 2009 When ammonium ion is limiting, most of glutamate is made by action of both enzymes to produce the following (sum of both reactions):

+ + NH4 +  -ketoglutarate + NADPH + ATP glutamate + NADP + ADP + Pi

Transamination Reactions

Having assimilated the ammonia, synthesis of nearly all amino acids is done via tranamination reactions. Glutamate is a key intermediate in amino acid metabolism Amino group is transferred to produce the corresponding  -amino acid.

transaminase <------>

-amino acid1 -keto acid -keto acid1 2 -amino acid2

Origins of Carbon Skeletons of the Amino Acids

Amino acids that must be supplied in diet are termed essential; others are nonessential. Although the biosynthesis of specific amino acids is diverse, they all share a common feature - carbon skeletons come from intermediates of glycolysis, PPP, or citric acid cycle. There are only six biosynthetic families: 1) Derived from oxaloacetate --> Asp, Asn, Met, Thr, Ile, Lys 2) Drived from pyruvate --> Ala, Val, Leu 3) Derived from ribose 5-phosphate --> His 4) Derived from PEP and erythrose 4-phosphate --> Phe, Tyr, Trp 5) Derived from a-ketoglutarate --> Glu, Gln, Pro, Arg 6) Derived from 3-phosphoglycerate --> Ser, Cys, Gly

Porphyrin Synthesis

First step in biosynthesis of porphyrins is condensation of glycine and succinyl CoA to form  -aminolevulinate via  -aminolevulinate synthase. Translation of mRNA of this enzyme is feedback-inhibited by heme Second step involves condensation of two molecules of  -aminolevulinate to form porphobilinogen; catalyzed by  -aminolevulinate dehydrase. 57 STUDY GUIDE for BIOCHEMISTRY 2009 Third step involves condensation of four porphobilinogens to form a linear tetrapyrrole via porphobilinogen deaminase. This is cyclized to form uroporphyrinogen III. Subsequent reactions alter side chains and degree of saturation of porphyrin ring to form protoporphyrin IX. Association of iron atom creates heme; iron atom transported in blood by transferrin.

Inherited or acquired disorders called porphyrias are result of deficiency in an enzyme in heme biosynthetic pathway. congenital erythropoietic porphyria - insufficient cosynthase (cyclizes tetrapyrrole) Lots of uroporphyrinogen I, a useless isomer are made RBCs prematurely destroyed Patient’s urine is red because of excretion of uroporphyrin I

Heme Degradation:

Old RBCs are removed from circulation and degraded by spleen. Apoprotein part of hemoglobin is hydrolyzed into amino acids. First step in degradation of heme group is cleavage of  -methene bridge to form biliverdin, a linear tetrapyrrole; catalyzed by heme oxygenase; methene bridge released as CO. Second step involved reduction of central methene bridge to form bilirubin; catalyzed by biliverdin reductase. Bilirubin is complexed with serum albumin --> liver --> sugar residues added to propionate side chains. 2 glucuronates attached to bilirubin are secreted in bile.

Jaundice - yellow pigmentation in sclera of eye and in skin --> excessive bilirubin levels in blood Caused by excessive breakdown of RBCs, impaired liver function, mechanical obstruction of bile duct. Common in newborns as fetal hemoglobin is broken down and replaced by adult hemoglobin.

AMINO ACID CATABOLISM

Excess amino acids (those not used for protein synthesis or synthesis of other macromolecules) cannot be stored. Surplus amino acids are used as metabolic fuel. -amino group is removed; carbon skeleton is converted into major metabolic intermediate Amino group converted to urea; carbon skeletons converted into acetyl CoA, acetoacetyl CoA, pyruvate, or citric acid intermediate. Fatty acids, ketone bodies, and glucose can be formed from amino acids. Major site of amino acid degradation is the liver. 58 STUDY GUIDE for BIOCHEMISTRY 2009 First step is the transfer of  -amino group to  -ketoglutarate to form glutamate, which is oxidatively deaminated to yield NH + (see pathway sheet). 4

Some of NH + is consumed in biosynthesis of nitrogen compounds; most 4

terrestrial vertebrates convert NH + into urea, which is then excreted 4 (considered ureotelic). Terrestrial reptiles and birds convert NH + into uric acid for excretion (considered 4 uricotelic). + Aquatic animals excrete NH (considered ammontelic). 4

In terrestrial vertebrates NH + is converted to urea via urea cycle. 4 One of nitrogen atoms in urea is transferred from aspartate; other is derived from + NH4 ; carbon atom comes from CO2.

UREA CYCLE

There are six steps of the urea cycle: + 1) Bicarbonate ion, NH4 and 2 ATP necessary to form carbamoyl phosphate via carbamoyl phosphate synthetase I (found in mitochondrial matrix). 2) Carbamoyl phosphate and ornithine (carrier or carbon and nitrogen atoms; an amino acid, but not a building block of proteins) combine to form citrulline via ornithine transcarbamoylase 3) Citruilline is transported out of mitochondrial matrix in exchange for ornithine 4) Citruilline condenses with aspartate --> arginosuccinate via an ATP- dependent reaction via arginosuccinate synthetase 5) Arginosuccinate cleaved to form fumarate and arginine via arginosuccinate lyase fumarate --> malate--> oxaloacetate --> gluconeogenesis oxaloacetate has four possible fates: 1) transamination to aspartate 2) conversion into glucose via gluconeogenesis 3) condensation with acetyl CoA to form citrate 4) conversion into pyruvate 6) Two -NH2 groups and terminal carbon of arginine cleaved to form ornithine and urea via arginase Ornithine is transported into mitochondrion to repeat cycle

Overall reaction:

+ CO2 + NH4 + 3 ATP + aspartate + 2 H2O ---> urea + 2 ADP + 2 Pi + AMP + PPi + fumarate

Inherited defects in urea cycle: 1) Blockage of carbamoyl phosphate synthesis leads to hyperammonemia (elevated levels of ammonia in blood) 2) argininosuccinase deficiency Providing surplus of arginine in diet and restricting total protein intake Nitrogen is excreted in the form of argininosuccinate 3) carbamoyl phosphate synthetase deficiency or ornithine transcarbamoylase deficiency Excess nitrogen accumulates in glycine and glutamine; must then get rid of these amino acids Done by supplementation with benzoate and phenylacetate (both substitute for urea in the disposal of nitrogen) benzoate --> benzoyl CoA --> hippurate phenylacetate --> phenylacetyl CoA --> phenylacetylglutamine

Fate of Carbon Skeleton of Amino Acids

Used to form major metabolic intermediates that can be converted into glucose or oxidized by citric acid cycle. All 20 amino acids are funneled into seven molecules: 1) pyruvate 2) acetyl CoA 3) acetoacetyl CoA 4) -ketoglutarate 5) succinyl CoA 6) fumarate 7) oxaloacetate Those that are degraded to acetyl CoA or acetoacetyl Coa are termed ketogenic because they give rise to ketone bodies. Those that are degraded to pyruvate or citric acid cycle intermediates are termed glucogenic. Leucine and lysine are only ketogenic --> cannot be converted to glucose Isoleucine, phenylalanine, tryptophan, tyrosine are both. All others are glucogenic only.

C3 family (alanine, serine, cysteine) ---> pyruvate C4 family(aspartate and asparagine) ---> oxaloacetate C5 family (glutamine, proline, arginine, histidine) ---> glutamate --->  - ketoglutarate Methionine, isoleucine, valine, threonine --> succinyl CoA Leucine --> acetyl CoA and acetoacetate 60 STUDY GUIDE for BIOCHEMISTRY 2009 Phenylalanine and tyrosine --> acetoacetate and fumarate Tryptophan --> pyruvate

Regulation of the Urea Cycle

The main allosteric enzyme is glutamate dehydrogenase. It is inhibited by high GTP and ATP levels. It is stimulated by high GDP and ADP levels.

Phenylketonuria

Phenylketonuria is (at least among Europeans) the most common hereditary enzyme defect. It is clinically manifest in about one among ten thousand persons. Considering that only homozygous people are clinically affected, this works out to a heterozygote frequency of (4×1/10,000)½ = 1/50, i.e. one in fifty persons can potentially have children with this disease. The enzyme affected is phenylalanine hydroxylase, the first enzyme in the degradative pathway . The name of the disease stems from the fact that phenylpyruvate and some derivatives thereof are found in the urine. Formation of phenylpyruvate is due to the buildup of phenylalanine, which will eventually cause it to overcome the low KM of tyrosine transaminase . Phenylpyruvate is believed to give rise to neurotoxic metabolites, although the exact nature of these metabolites remains to be elucidated. Symptoms include disturbances in neurological development and mental retardation. The treatment of phenylketonuria is pretty straightforward: Limitation of dietary phenylalanine. Tyrosine is plentifully available in a modern, protein-rich diet, so that the lack of endogenous formation won’t be a problem. The challenge is then to diagnose the disease in newborn kids, before any damage is done. Happily, the enzyme defect does not cause a problem during fetal development, since both useful and potentially harmful metabolites are constantly equilibrated between the maternal and the fetal circulation. Buildup of a metabolite in the fetus will therefore not occur as long as the mother’s metabolism is able to degrade it.

CHAPTER 6: CARBOHYDRATE 61 STUDY GUIDE for BIOCHEMISTRY 2009

Carbohydrates: Bountiful Sources of Energy and Nutrients What Are Carbohydrates? ♦One of the three macronutrients ♦Preferred energy source for the brain ♦Important source of energy for all cells ♦Composed of carbon, hydrogen, oxygen ♦Good sources: fruits, vegetables, and grains Simple carbohydrates � Contain one or two molecules � Commonly referred to as sugars Monosaccharides contain only one molecule � Glucose, Fructose, Galactose Disaccharides contain two molecules � Lactose, Maltose, Sucrose Complex carbohydrates � Long chains of glucose molecules � Starch, fiber, glycogen Simple Carbohydrates – Monosaccharides Glucose Fructose Galactose

Simple Sugars – Dissacharides

Complex Carbohydrates � Long chains of glucose molecules � Hundreds to thousands of molecules long � Also called polysaccharides � Starch, glycogen, most Fibers

Complex Carbohydrates

63 STUDY GUIDE for BIOCHEMISTRY 2009 Starch �Plants store carbohydrates as starch �We digest (break down) starch to glucose �Good sources: grains, legumes, and Tubers

Glycogen � Animals store carbohydrates as glycogen � Stored in the liver and muscles � Not found in food and therefore not a source of dietary carbohydrate

Fiber � Dietary fiber is the non-digestible part of plants � Grains, seeds, legumes, fruits � Functional fiber is carbohydrate extracted from plants or manufactured � Total fiber = dietary + functional fiber � Food labels only list dietary fiber

Salivary amylase � Enzyme that begins carbohydrate digestion in the mouth � Breaks carbohydrates down to maltose Carbohydrate digestion does not occur in the stomach. Stomach acids inactivate salivary amylase Most chemical digestion of carbohydrates occurs in the small intestine. Pancreatic amylase � Enzyme produced in the pancreas and secreted into the small intestine Digests carbohydrates to maltose

Additional enzymes in the small intestine digest disaccharides to monosaccharides � Maltase – breaks down maltose into two units of glucose � Sucrase – breaks down sucrose into glucose & fructose � Lactase – breaks down lactose into glucose & galactose Monosaccharides are absorbed into the cells lining the small intestine and then enter the bloodstream.

All monosaccharides are converted to glucose by the liver. Glucose circulating in the blood is our primary energy source. Excess glucose is converted to glycogen by the LIVER We do not have the enzymes necessary to digest fiber. Bacteria in the large intestine can break down (ferment) some fiber. Most fiber remains undigested and is excreted in the faeces

Glucose Utilization

Blood Glucose Regulation Blood glucose level must be closely regulated. Hormones control blood glucose levels: � Insulin � Glucagon � Epinephrine � Norepinephrine � Cortisol Growth hormone Blood Glucose Regulation Insulin Produced by beta cells of the pancreas Stimulates glucose transporters (carrier proteins) to help take glucose from the blood across the cell membrane Stimulates the liver to take up glucose and convert to glycogen

Blood Glucose Regulation Glucagon Produced by alpha cells of the pancreas Stimulates the liver to breakdown glycogen to glucose, making glucose available to body cells Stimulates the breakdown of body proteins to amino acids to form new glucose - Gluconeogenesis

TUTORIAL 4, ENV416/400

A. Carbohydrates

1. Name the monosaccarides produced from the hydrolysis of the dissaccharides below:

i) Sucrose 67 STUDY GUIDE for BIOCHEMISTRY 2009 ii) Lactose

2. Explain the difference between glucose and fructose in terms of their structure.

3. D-Allosa, an aldohexose, has the same structure as D-glucose except that the carboxyl at C3 is at the plane below in the cyclic hemiacetyl form. Draw the structure of β-cyclic for D-allosa.

4. Name the two component of starch. State the similarity and difference between these two structures.

5. Draw the open chain structure (Fischer projection) for D- fructose. Indicate the carbonyl ketone or aldehyde group on the molecule.

6. Draw the cyclic β-D-fructose (Haworth structure).

B. Nitrogen Metabolism

1. Write the transamination reaction between α-ketoglutarate and alanine.

2. Write an equation for the net reaction of the urea cycle. Show how the urea cycle is linked to the citric acid cycle.

3. Which aa in the urea cycle are the links to the citric acid cycle? Show how these links occur.

4. How many ATP’s are required for one round of the urea cycle? Where do these ATPs get used?

5. What species excrete excess nitrogen as ammonia? Which ones excrete as uric acid?

CHAPTER 7 :Metabolic processes central to ATP synthesis- Glycolysis and Citric acid cycle

Glycolysis

Purpose: catabolism of glucose to provide ATPs and NADH molecules Also provides building blocks for anabolic pathways. Sequence of 10 enzyme-catalyzed reactions:

glucose pyruvate 2 ATPs and 2 NADH produced

All enzymes (and reactions) are cytosolic. Net reaction:

+ glucose + 2ADP + 2NAD +2Pi 2 pyruvate + 2ATP + 2NADH + +2H +2H2O

Can catabolize sugars other than glucose:

e.g. fructose ----> 2 glyceraldehyde 3-phosphate e.g. lactose --> glucose + galactose galactose --> glucose 1-phosphate --> glucose 6-phosphate e.g. mannose ---> mannose 6-phosphate --> fructose 6-phosphate

Ten Steps of Glycolysis

1) glucose --> glucose 6-phosphate by hexokinase G = -8.0 kcal/mole

Hexokinase also works on mannose and fructose at increased [ ]. Serves to trap glucose in the cell --> a phosphorylated molecule cannot leave

2) glucose 6-phosphate --> fructose 6-phosphate by glucose 6-phosphate isomerase Example of aldose--> ketose isomerization. Enzyme is very stereospecific. Reaction is near equilibrium in cell --> not a control point in glycolysis

3) fructose 6-phosphate --> fructose 1,6-bisphosphate by phosphofructokinase-1 (PFK-1) Reaction has G = -5.3 kcal/mole and is metabolically irreversible. Represents the first committed step in glycolysis.

69 STUDY GUIDE for BIOCHEMISTRY 2009 4) fructose 1,6-bisphosphate --> dihydroxyacetone phosphate + glyceraldehyde 3-phosphate by fructose 1,6 bisphosphate aldolase.

5) DHAP --> glyceraldehyde 3-phosphate by triose phosphate isomerase Also catalyzes aldose--> ketose conversion. Rate is diffusion controlled (substrate is converted to product as fast as substrate is encountered).

6) glyceraldehyde 3-phosphate --> 1,3-bisphosphoglycerate by glyceraldehyde 3- phosphate dehydrogenase + One molecule of NAD is reduced to NADH --> respiratory chain

7) 1,3 bisphosphoglycerate --> 3-phosphoglycerate Phosphoryl group transfer to ADP to form ATP. Because phosphate group comes from a substrate molecule, called substrate level phosphorylation First ATP-generating step of glycolysis.

8) 3-phosphoglycerate --> 2-phosphoglycerate by phosphoglycerate mutase Mutases are enzymes that transfer phosphoryl groups from one part of a substrate molecule to another.

9) 2-phosphoglycerate --> phosphoenolpyruvate (PEP) by enolase (forms double bond)

10) PEP --> pyruvate Second time for substrate level phosphorylation. Reaction is metabolically irreversible.

FATE OF PYRUVATE

Under anaerobic conditions, cells must be able to regenerate NAD+ or glycolysis will stop. Usually regenerated by oxidative phosphorylation, but that requires O2. + There are 2 anaerobic pathways that use NADH and regenerate NAD . 1) alcoholic fermentation Conversion of pyruvate to ethanol

+ + H CO2 NADH NAD pyruvate acetaldehyde ethanol pyruvate alcohol

70 STUDY GUIDE for BIOCHEMISTRY 2009 decarboxylase dehydrogenase

+ glucose +2Pi + 2ADP + 2H ---> 2 ethanol + 2CO2 + 2ATP + 2H2O

2) lactate fermentation

NADH + H+ NAD+ pyruvate ------> lactate lactate dehydrogenase

glucose +2Pi + 2ADP ---> 2 lactate + 2ATP + 2H20

Lactate causes muscles to ache. Also produced by bacterial fermentation of lactose.

3) entry into citric acid cycle

The Citric Acid Cycle Summary:

Yields reduced coenzymes (NADH and QH2) and some ATP (2). Preparative step is oxidative decarboxylation involving coenzyme A. Occurs in eucaryotic mitochondrion and procaryotic cytosol.

How does the pyruvate get into the mitochondrion from the cytosol? Pyruvate passes through channel proteins called porins (can transport molecules < 10,000 daltons) located in outer mitochondrial membrane. To get from intermembrane space to matrix involves pyruvate translocase (symporter that also moves H+ into matrix).

CONVERSION OF PYRUVATE TO ACETYL COA Enzyme is pyruvate dehydrogenase complex, composed of three enzymes: 1) pyruvate dehydrogenase 2) dihydrolipoamide acetyltransferase 3) dihydrolipoamide dehydrogenase

71 STUDY GUIDE for BIOCHEMISTRY 2009 Reaction occurs in 5 steps:

1) E1 uses TPP as a prosthetic group and decarboxylates pyruvate --> forms HETPP intermediate

2) E1 then transfers acetyl group to oxidized lipoamide --> acetyllipoamide

3) E2 transfers acetyl group to coenzyme A to form acetyl CoA; dihydrolipoamide becomes reduced

4) E3 reoxidizes lipoamide portion of E2; prosthetic group of E3 (FAD) oxidizes reduced lipoamide --> FADH2 + + 5) NAD is reduced by E3-FADH --> E3-FAD + NADH + H

E2 acts like a crane by swinging substrate between protein complexes in enzyme.

Regulation of PDH complex:

Regulated by covalent modification by phosphorylation. inactive = phosphorylated; active = dephosphorylated

E1 inhibited at high [ATP]; inhibited at high [GTP] activated by high [AMP], high [Ca2+], high [pyruvate]

E2 inhibited by high [acetyl CoA] activated by high [CoA-SH]

E3 inhibited by high [NADH] activated by high [NAD+]

THE CITRIC ACID CYCLE

Summary: Composed of 8 reactions 4 carbon intermediates are regenerated

2 molecules of CO2 released (6C--> 4C)

Most of energy stored as NADH and QH2 1) citrate synthase Irreversible reaction Acetyl CoA reacts with oxaloacetate --> citrate and CoA

2) aconitase Citrate --> isocitrate

3) isocitrate dehydrogenase Irreversible reaction Substrate first oxidized (2e- and H+ given to NAD+), then decarboxylated + Isocitrate --> -ketoglutarate + CO2 + NADH + H

4) -ketoglutarate dehydrogenase complex -ketoglutarate first decarboxylated, oxidized (2e- and H+ given to NAD+), and HS-CoA added Product is succinyl CoA Enzyme complex similar the PDH, but has dihydrolipoamide succinyltransferase instead of acetyltransferase.

5) succinyl CoA synthetase or succinate thiokinase succinyl CoA --> succinate Substrate has high energy thioester bond; that energy is stored as nucleoside triphosphate via substrate level phosphorylation

GDP +Pi --> GTP mammals

ADP +Pi --> ATP plants and bacteria

6) succinate dehydrogenase complex Enzyme is embedded in inner mitochondrial membrane. Has FAD covalently bound to it (prosthetic group).

Converts succinate --> fumarate with generation of FADH2 --> ETS FAD is regenerated by reduction of a mobile molecule called ubiquinone (coenzyme Q) --> QH2.

CHAPTER 8 :Electron Transport and Oxidative Phosphorylation 73 STUDY GUIDE for BIOCHEMISTRY 2009

 Oxidative phosphorylation - process in which NADH and QH2 are oxidized and ATP is produced.  Enzymes are found in inner mitochondrial membrane in eukaryotes.  In prokaryotes, enzymes are found in cell membrane.  Process consists of 2 separate, but coupled processes: 1) respiratory electron-transport chain

 Responsible for NADH and QH2 oxidation -  Final e acceptor is molecular oxygen  Energy generated from electron transfer is used to + pump H into intermembrane space from matrix ---> matrix becomes more alkaline and negatively charged. 2) ATP synthesis  Proton concentration gradients represents stored energy  When H+ are moved back across inner mitochondrial membrane through ATP synthase ---> ADP is phosphorylated to form ATP

Chemiosmotic Theory of ATP Production

Proposed by Peter Mitchell in 1961 (won Nobel Prize for this work). Proton concentration gradient serves as energy reservoir for ATP synthesis. Proton concentration gradient also known as proton motive force (PMF).

Components of Electron Transport System

There are 5 protein complexes: I) NADH-ubiquinone oxidoreductase II) succinate-ubiquinone oxidoreductase III) ubiquinol-cytochrome c oxidoreductase IV) cytochrome c oxidase V) ATP synthase

 Electrons flow through ETS in direction of increasing reduction potential.  Two mobile electron carriers also involved: ubiquinone (Q) between complexes I or II and III, and cytochrome c between complexes III and IV.  Electrons enter ETS 2 at a time from either NADH or succinate.

I - NADH-ubiquinone oxidoreductase - Transfers 2e- from NADH to Q as hydride ion (H ) 74 STUDY GUIDE for BIOCHEMISTRY 2009 First electron transferred to FMN --> FMNH2 ---> Fe-S cluster ---> Q Also pumps 4H+/2e- into intermembrane space

II - succinate-ubiquinone oxidoreductase Transfers e- from succinate to Q First transferred to FAD ---> FADH2 ---> 3 Fe-S clusters ---> Q Not enough energy to contribute to proton gradient via proton pumping

III - ubiquinol-cytochrome c oxidoreductase - Rransfers e from QH2 to cytochrome c facing intermembrane space Composed of 9-10 subunits including 2 Fe-S clusters, cytochrome b560, cytochrome b566, and cytochrome c1.

Transports 2H+ from matrix into intermembrane space

IV - cytochrome c oxidase Contains cytochromes a and a3 Contributes to proton gradient in two ways: + - 1) pumps 2H for each pair of e transferred (per O2 reduced) + + 2) consumes 2H when oxygen is reduced to H2O ---> lowers [H ]matrix Carbon monoxide (CO) and cyanide (HCN) bind here

V - ATP synthase Does not contribute to H+ gradient, but helps relieve it Also called FOF1 ATP synthase F component contains catalytic subunits 1 F component is proton channel that is transmembrane O Per ATP synthesized, 3H+ move through ATP synthase oligomycin - antibiotic that binds to channel and prevents proton entry --> no ATP synthesized

TRANSPORT OF MOLECULES ACROSS MITOCHONDRIAL MEMBRANE

 Inner mitochondrial membrane is impermeable to NADH and NAD+.  Must use a shuttle to regenerate NAD+ for glycolysis; solution is to shuttle electrons across membrane, rather than NADH itself.  There are two shuttles in operation: 1) glycerol phosphate shuttle . Found in insect flight muscles and mammalian cells in which high rates of oxidative phosphorylation must occur . Cytosolic glycerol 3-phosphate dehydrogenase converts DHAP to glycerol 3-phosphate . Converted back to DHAP by membrane-bound glycerol 3- phosphate dehydrogenase . Result is transfer to 2e- to FAD --> Q ---> complex III

75 STUDY GUIDE for BIOCHEMISTRY 2009 . Produces fewer ATP molecules (1.5 vs. 2) because complex I is bypassed 2) malate-aspartate shuttle  Found in liver and heart  Cytosolic NADH reduces oxaloacetate --> malate --> transported via dicarboxylate translocase into matrix  In matrix, malate --> oxaloacetate --> aspartate ---> transported out via glutamate-aspartate translocase  Converted back to oxaloacetate......  No reduction in ATP yield

Must also be able to transport other metabolites into and out of matrix: 1) ADP/ATP carrier or ADP/ATP translocase Adenine nucleotide translocase which exchanges ADP and ATP (antiporter) + 2) Pi/H carrier + Couples inward movement of Pi with symport of H from gradient

REGULATION OF OXIDATIVE PHOSPHORYLATION

 Depends upon substrate availability and energy demands in the cell.

 Important substrates are NADH, O2, and ADP.  As ATP is used, more ADP is available, translocated through adenine nucleotide translocase --> electron transport increases.  Known as respiratory control.  Helps to replenish ATP pool in the cell, which is kept nearly constant.  Rates of glycolysis, citric acid cycle, and electron transport system are matched to a cell’s ATP requirements.  Proton gradient can be short-circuited to generate heat  Found in brown adipose tissue in newborn mammals and animals that hibernate, and animals adapted to cold conditions  A protein called thermogenin forms a proton channel in inner mitochondrial membrane --> dissipates proton gradient, but electrons still flow --> heat production  Pathway is activated by fatty acids from triacylglycerol catabolism from epinephrine stimulation

Superoxide Production

Even though cytochrome oxidase and other proteins that reduce oxygen have .- been designed not to release O2 (superoxide anion), it still does happen.

76 STUDY GUIDE for BIOCHEMISTRY 2009 . Protonation of superoxide anion yields hydroperoxyl radical (HO2 ), which can react with another molecule to produce H2O2. Enzyme superoxide dismutase catalyzes this reaction 2H+ .- .- O2 + O2 ------> H2O2 + O2 superoxide dismutase

Recent findings have indicated that superoxide dismutase mutations can cause amyotrophic lateral sclerosis (Lou Gehrig’s disease), in which motor neurons in brain and spinal cord degenerate.

The hydrogen peroxide formed is scavenged by catalase:

H O + H O 2H O + O 2 2 2 2 2 2 catalase

Peroxidases catalyze an analogous reaction:

ROOH + AH2 ROH + H2O + A peroxidase 7) fumarase fumarate --> malate

8) malate dehydrogenase L-malate --> oxaloacetate 2e- and H+ given to NAD+ --> NADH

Net reaction for citric acid cycle:

acetyl CoA + 3NAD+ + Q + GDP(ADP)+ Pi +2H2O ---> HS-CoA + 3NADH + QH2 + GTP(ATP) + + 2CO2 + 2H Energy Budget so far from 1 molecule of glucose:

glycolysis 2 ATP 2 NADH Prep Step 2 NADH

TCA 2 ATP 6 NADH 2 QH2 77 STUDY GUIDE for BIOCHEMISTRY 2009 4 ATP 10 NADH ATP Production:

glycolysis 2 ATP 6 ATP equivalents Prep Step 6 ATP equivalents TCA 2 ATP 18 ATP equivalents + 4 ATP equivalents

4 ATP 34 ATP = 38 ATPs maximum substrate (ox. phos.) level phos.

REGULATION OF TCA CYCLE

There are 2 enzymes that are regulated:

1) isocitrate dehydrogenase allosterically activated by high [Ca2+] and high [ADP] allosterically inhibited by high [NADH]

2) -ketoglutarate dehydrogenase allosterically activated by high [Ca2+] allosterically inhibited by high [NADH] and high [succinyl CoA]

ENTRY AND EXIT OF METABOLITES

Citrate, -ketoglutarate, succinyl CoA, oxaloacetate lead to biosynthetic pathways.

Citrate --> fatty acids and sterols in liver and adipocytes 78 STUDY GUIDE for BIOCHEMISTRY 2009 (cleaved into acetyl CoA if needed)

-ketoglutarate --> glutamate --> amino acid synthesis or nucleotide synthesis succinyl CoA --> propionyl CoA --> fatty acid synthesis --> porphyrin synthesis oxaloacetate --> gluconeogenesis --> asparate --> urea synthesis, a.a. synthesis, pyrimidine synthesis

Pathway intermediates must be replenished by anapleurotic reactions.

GLYOXYLATE CYCLE Modification of citric acid cycle. Anabolic pathway in plants, bacteria, yeast. Takes 2 carbon compounds and converts them to glucose. Common in plants which store energy reserves as oils, but must be converted to carbohydrates during germination. In eucaryotes, a glyoxysome is a special organelle where this occurs.

Gluconeogenesis, the Pentose Phosphate Pathway and Glycogen Metabolism

GLYCOGEN METABOLISM

 Glycogen stored in muscle and liver cells.  Important in maintaining blood glucose levels.  Glycogen structure:  1,4 glycosidic linkages with  1,6 branches.  Branches give multiple free ends for quicker breakdown or for more places to add additional units. Glycogen Degradation

 Glucose residues of starch and glycogen released through enzymes called starch phosphorylases and glycogen phosphorylases.  Catalyze phosphorolosis:

79 STUDY GUIDE for BIOCHEMISTRY 2009 polysaccharide +Pi ---> polysaccharide(n-1) + glucose 1-phosphate

 Pyridoxal phosphate (PLP) is prosthetic group in active site of enzyme; serves as a proton donor in active site.  Allosterically inhibited by high [ATP] and high [glucose 6-phosphate].  Allosterically activated by high [AMP].  Sequentially removes glucose residues from nonreducing ends of glycogen, but stops 4 glucose residues from branch point --> leaves a limit dextran.  Limit dextran further degraded by glycogen-debranching enzyme (glucanotransferase activity) which relocated the chain to a free hydroxyl end.  Amylo-1,6-glucosidase activity of debranching enzyme removes remaining residues of chain.  This leaves substrate for glycogen phosphorylase.  Each glucose molecule released from glycogen by debranching enzyme will yield 3 ATPs in glycolysis.  Each glucose molecule released by glycogen phosphorylase will yield 2 ATPs in glycolysis.  Why? . ATP not needed in first step because glucose 1-phosphate already formed.

phosphoglucomutase glucose 1-phosphate ------> glucose 6-phosphate

1) In liver, kidney, pancreas, small intestine,

glucose 6-phosphatase

glucose 6-phosphate ------> glucose + Pi Glycogen Synthesis  Not reverse of glycogen degradation because different enzymes are used.  About 2/3 of glucose ingested during a meal is converted to glycogen.  First step is the first step of glycolysis:

hexokinase glucose ------> glucose 6-phosphate

 There are three enzyme-catalyzed reactions:

phosphoglucomutase glucose 6-phosphate ------> glucose 1-phosphate

glucose 1-phosphate ------> UDP-glucose (activated form of glucose)

glycogen synthase UDP-glucose ------> glycogen

 Glycogen synthase cannot initiate glycogen synthesis; requires preexisting primer of glycogen consisting of 4-8 glucose residues with  (1,4) linkage.  Protein called glycogenin serves as anchor; also adds 7-8 glucose residues.  Addition of branches by branching enzyme (amylo-(1,4 --> 1,6)- transglycosylase).  Takes terminal 6 glucose residues from nonreducing end and attaches it via (1,6) linkage at least 4 glucose units away from nearest branch.

REGULATION OF GLYCOGEN METABOLISM

Mobilization and synthesis of glycogen under hormonal control.

Three hormones involved:

1) insulin  51 a.a. protein made by  cells of pancreas.  Secreted when [glucose] high --> increases rate of glucose transport into muscle and fat via GLUT4 glucose transporters.  Stimulates glycogen synthesis in liver.

2) glucagon  29 a.a. protein secreted by  cells of pancreas.  Operational under low [glucose].  Restores blood sugar levels by stimulating glycogen degradation.

3) epinephrine

81 STUDY GUIDE for BIOCHEMISTRY 2009  Stimulates glycogen mobilization to glucose 1-phosphate --> glucose 6- phosphate.  Increases rate of glycolysis in muscle and the amount of glucose in bloodstream.  Occurs in response to fight-or-flight response.

 Binds to -adrenergic receptors in liver and muscle and 1 receptors in liver cells.  Binding of epinephrine or glucagon to  receptors activates adenylate cyclase, which is a membrane-traversing enzyme that converts ATP --> cAMP --> activates protein kinase A.

 Binding of epinephrine to 1 receptors activates IP3 pathway --> protein kinase C --> phosphorylation of insulin receptors -> insulin cannot bind.

Regulation of glycogen phosphorylase and glycogen synthase  Reciprocal regulation. . Glycogen synthase -P --> inactive form (b). . Glycogen phosphorylase-P ---> active (a).  When blood glucose is low, protein kinase A activated through hormonal action of glucagon --> glycogen synthase inactivated and phosphorylase kinase activated --> activates glycogen phosphorylase --> glycogen degradation occurs.  Phosphorylase kinase also activated by increased [Ca2+] during muscle contraction.  To reverse the same pathway involves protein phosphatases, which remove phosphate groups from proteins --> dephosphorylates phosphorylase kinase and glycogen phosphorylase (both inactivated), but dephosphorylation of glycogen synthase activates this enzyme.  Protein phosphatase-1 activated by insulin --> dephosphorylates glycogen synthase --> glycogen synthesis occurs.  In liver, glycogen phosphorylase a inhibits phosphatase-1 --> no glycogen synthesis can occur.  Glucose binding to protein phosphatase-1 activated protein phosphatase-1 --> it dephosphorylates glycogen phosphorylase --> inactivated --> no glycogen degradation.  Protein phosphatase-1 can also dephosphorylate glycogen synthase --> active.

GLUCONEOGENESIS  Synthesis of glucose from noncarbohydrate sources.  Major precursors are lactate and alanine in the liver and kidney.  lactate - active skeletal muscles  glycerol - lipid catabolism

82 STUDY GUIDE for BIOCHEMISTRY 2009  amino acids - diet and protein catabolism  Used to maintain blood glucose levels when glycogen supplies are low or depleted.  Major site of occurrence is the liver, but also occurs in kidney.  Designed to make sure blood glucose levels are high enough to meet the demands of brain and muscle (cannot do gluconeogenesis).  NOT the reverse of glycolysis. Why?  PFK, PK, and hexokinase catalyze metabolically irreversible steps.  Solution: by-pass these steps, but use all the other enzymes.

1) pyruvate ---> phosphoenolpyruvate

ATP ADP + Pi GTP GDP pyruvate ------> oxaloacetate ------> PEP - HCO3 pyruvate PEP carboxykinase carboxylase TCA Cycle

Pi 2) fructose 1,6 bisphosphate fructose 6-phosphate

fructose 1,5-bisphosphatase

glucose 6-phosphatase 3) glucose 6-phosphate ------> glucose

This enzyme is bound to ER membrane, but faces ER lumen. GLUT7 transporter must transport glucose 6-phosphate into ER lumen. Enzyme not found in membrane of brain or muscle

PRECURSORS FOR GLUCONEOGENESIS

1) lactate Cori cycle - no net gain or loss of glucose Anaerobic respiration of pyruvate.

2) amino acids glutamate -ketoglutarate

pyruvate ------> alanine transamination

3) glycerol glycerol kinase glycerol ------> glycerol 3-phosphate -----> DHAP If glycerol 3-phosphate dehydrogenase is embedded in inner mitochondrial membrane, e- passed to ubiquinone. If enzyme is cytosolic, NADH is also a product.

REGULATION OF GLUCONEOGENSIS

 Glycolysis and gluconeogenesis are reciprocally regulated.  If both pathways were activated, e.g.

fructose 6-phosphate + ATP ------> fructose 1,6-bisphosphate + ADP

fructose 1,6-bisphosphate + H2O ---> fructose 6-phosphate + Pi

net reaction: ATP + H2O ---> ADP + Pi

 Called substrate cycle ---> “burn” 4 ATPs for every 2 ATPs made (can be used to generate heat).  Reason why enzymes are regulated --> prevents this from happening.

 Two regulatory points are the two steps which had different enzymes. fructose 1,6-bisphosphatase inhibited by AMP and fructose 2,6-bisphosphate pyruvate carboxylase activated by acetyl CoA

PENTOSE PHOSPHATE PATHWAY

Provides NADPH (serves as e- donor) and forms ribose 5-phosphate (nucleotide synthesis). Pathway active is tissues that synthesize fatty acids or sterols because large amounts of NADPH needed. In muscle and brain, little PPP activity. All reactions are cytosolic. Divided into 2 stages: 1) oxidative + glucose 6-phosphate +2 NADP + H2O --> ribulose 5-phosphate + + 2 NADPH + CO2 + 2H 2) nonoxidative Uses transketolases (transfers 2-C units) and transaldolases (transfers 3-C units). Links PPP with glycolysis. Used to catalyze these types of reactions: C5 + C5 <----> C7 + C3 C7 + C3 <----> C4 + C6 C5 + C4 <----> C3 + C6 All reactions are reversible --> very flexible pathway. Example:

85 STUDY GUIDE for BIOCHEMISTRY 2009  If ribose 5-phosphate needed, fructose 6-phosphate + glyceraldehyde 3- phosphate taken from glycolysis and channeled through PPP to make product.  If NADPH is needed, then ribulose 5-phosphate is converted to glyceraldehyde 3-phosphate and fructose 6-phosphate --> converted to glucose 6-phosphate --> more NADPH made.  If use PPP, 1 glucose can be completely oxidized to 12 NADPH and 6 CO2.  If NADPH and ATP are needed, ribulose 5-phosphate converted into glyceraldehyde 3-phosphate and fructose 6-phosphate --> glycolysis --> pyruvate.

REGULATION OF PENTOSE PHOSPHATE PATHWAY

 Controlled by levels of NADP+.  Controlled step is dehydrogenation of glucose 6-phosphate to 6- phosphogluconolactone.  Enzyme stimulated by high [NADP+].  Nonoxidative branch controlled primarily by substrate availability.

Health Disorders

Three health disorders related to carbohydrate metabolism are � Diabetes � Hypoglycemia� Lactose intolerance

Diabetes Inability to regulate blood glucose levels Three types: � Type 1 diabetes � Type 2 diabetes � Gestational diabetes Uncontrolled diabetes can cause nerve damage, kidney damage, blindness, and can be fatal Diabetes – Type 1 Accounts for 10% of all cases Patients do not produce enough insulin Causes hyperglycemia (high blood glucose) Requires insulin injections May be an autoimmune disease Once known as juvenile-onset diabetes or insulin dependent diabetes mellitus (IDDM)

86 STUDY GUIDE for BIOCHEMISTRY 2009 Diabetes – Type 2 Most diabetics have type 2 diabetes Progressive disease with biological changes occurring over time Body cells become resistant, or less responsive to insulin Hyperglycemia results when cells cannot take in the glucose from the blood Once known as non-insulin dependent diabetes mellitus (NIDDM) Diabetes – Type 2 Cause is unclear but genetics, obesity, and physical inactivity play a large role Treat with weight-loss diet, regular exercise, and, if necessary, medications Healthy lifestyle choices may prevent or delay the onset of type 2 diabetes: � Balanced diet and regular exercise � Achieving and maintaining healthy body weight Hypoglycemia Low blood sugar (glucose) Causes shakiness, sweating, anxiety Reactive hypoglycemia: pancreas secretes too much insulin after a high- carbohydrate meal Fasting hypoglycemia: pancreas produces too much insulin, even when someone has not eaten.

Lactose Intolerance Insufficient enzyme lactase to digest the lactose containing foods Symptoms: gas, bloating, cramping, diarrhea Extent of intolerance: mild to severe Persons with lactose intolerance may need to find alternate sources of calcium CHAPTER 9 :Lipids and Membranes Lipids are water-insoluble that are either hydrophobic (nonpolar) or amphipathic (polar and nonpolar regions). There are many types of lipids: 1) fatty acids  The simplest with structural formula of R-COOH where R = hydrocarbon chain.  They differ from each other by the length of the tail, degree of unsaturation, and position of double bonds.

 pKa of -COOH is 4.5-5.0 --> ionized at physiological pH.  If there is no double bond, the fatty acid is saturated.  If there is at least one double bond, the fatty acid is unsaturated.  Monounsaturated fatty acids contain 1 double bond; polyunsaturated fatty acids have >2 double bonds.  IUPAC nomenclature =n represents where double bond occurs as you count from the carboxyl end (see Table 9.1). e.g. -enoate one double bond

87 STUDY GUIDE for BIOCHEMISTRY 2009 -dienoate 2 “ -trienoate 3 “ -tetraenoate 4 “  Can also use a colon separating 2 numbers, where the first number represents the number of carbon atoms and the second number indicates the location of the double bonds. e.g. linoleate 18:29,12 or cis,cis -9,12octadecadienoate  Physical properties differ between saturated and unsaturated fatty acids. Saturated = solid at RT; often animal source; e.g. lard Unsaturated = liquid at RT; plant source; e.g. vegetable oil  The length of the hydrocarbon tails influences the melting point.  As the length of tails increases, melting points increases due to number of van der Waals interactions.  Also affecting the melting point is the degree of unsaturation.  As the degree of unsaturation increases, fatty acids become more fluid--> melting point decreases ( kinks in tails decrease number of van der Waals interactions).  Fatty acids are also an important sources of energy. 9 kcal/g vs. 4 kcal/g for carbohydrates and proteins.

2) triacylglycerols

 Also called triglycerides.  Made of 3 fatty acyl residues esterified to glycerol.  Very hydrophobic, neutral in charge ---> can be stored in anhydrous form.  Long chain, saturated triacylglycerols are solid at RT (fats).  Shorter chain, unsaturated triacylglycerols are liquid at RT (oils).  Lipids in our diet are usually ingested as triacylglycerols and broken down by lipases to release fatty acids from their glycerol backbones  Also occurs in the presence of detergents called bile salts. . Form micelles around fatty acids that allow them to be absorbed by intestinal epithelial cells. . Transported through the body as lipoproteins.

3) glycerophospholipids  Main components of cell membranes.  Are amphipathic and form bilayers.  All use glycerol 3-phosphate as backbone.  Simplest is phosphatidate = 2 fatty acyl groups esterified to glycerol 3- phosphate. 88 STUDY GUIDE for BIOCHEMISTRY 2009  Often, phosphate is esterified to another alcohol to form... . phosphatidylethanolamine . phosphatidylserine . phosphatidylcholine

 Enzymes called phospholipases break down biological membranes. . A-1 = hydrolysis of ester bond at C-1. . A-2 = hydrolysis of ester bond at C-2; found in pancreatic juice. . C = hydrolysis of P-O bond between glycerol and phosphate to create phosphatidate. . D = same 4) sphingolipids  Second most important membrane constituent.  Very abundant in mammalian CNS.  Backbone is sphingosine (unbranched 18 carbon alcohol with 1 trans + C=C between C-4 and C-5), NH3 group at C-2, hydroxyl groups at C-1 and C-3.  Ceramides are intermediates of sphingolipid synthesis.  There are three families of sphingolipids: 1) sphingomyelin - phosphocholine attached to C-1 hydroxyl group of ceramide; present in the myelin sheaths around some peripheral nerves. 2) 2)cerebrosides - glycosphingolipid; has 1 monosaccharide (galactose) attached by  -glycosidic linkage to C-1 of ceramide; most common is galactocerebroside, which is abundant in nervous tissue. 3) gangliosides - glycosphingolipid containing N-acetylneuraminic acid; present on all cell surfaces.  Hydrocarbon tails embedded in membrane with oligosaccharides facing extracellularly.  Probably used as cell surface markers, e.g. ABO blood group antigens.  Inherited defects in ganglioside metabolism --> diseases, such as Tay- Sachs disease. 5) steroids  Called isoprenoids because their structure is similar to isoprene.  Have 4 fused rings: 3 6-membered rings (A,B,C) and 1 5-membered ring (D).  Cholesterol is an important component of cell membranes of animals, but rare in plants and absent in procaryotes.  Also have mammalian steroid hormones (estrogen, androgens) and bile salts.

89 STUDY GUIDE for BIOCHEMISTRY 2009  Differ in length of side chain at C-17, number and location of methyl groups, double bonds, etc.  Cholesterol’s role in membranes is to broaden the phase transition of cell membranes ---> increases membrane fluidity because cholesterol disrupts packing of fatty acyl chains.

6) other lipids not found in membranes  waxes - nonpolar esters of long chain fatty acids and alcohols very water insoluble high melting point --> solid at outdoor/RT. Roles: protective coatings of leaves, fruits, fur, feathers, exoskeletons.

 eicosanoids - 20 carbon polyunsaturated fatty acids e.g. prostaglandins - affect smooth muscle --> cause constriction; bronchial constriction of asthmatics; uterine contraction during labo  limonene - smell of lemons  bactoprenol - involved in cell wall synthesis  juvenile hormone I - larval development of insects

Biological Membranes  Central transport of ions and molecules into and out of the cell.  Generate proton gradients for ATP production by oxidative phosphorylation.  Receptors bind extracellular signals and transduce the signal to cell interior.  Structure:  Glycerophospholipids and glycosphingolipids form bilayers.  Noncovalent interactions hold lipids together.  5-6 nm thick and made of 2 leaflets to form a lipid bilayer driven by hydrophobic effects.  About 40% lipid and 50% proteins by mass, with about 10% carbohydrates.  Protein and lipid composition varies among membranes but all have same basic structure --> Singer and Nicholson fluid mosaic model in 1972. Membrane fluidity:  Lipids can undergo lateral diffusion; can move about 2  m/sec.  Can undergo transverse diffusion (one leaflet to another) but very rare.

90 STUDY GUIDE for BIOCHEMISTRY 2009  Membrane has an asymmetrical lipid distribution that is maintained by flippases or translocases that are ATP-driven.  In 1970, Frye and Edidin demonstrated that proteins are also capable of diffusion by using heterocaryons, but occurs at a rate that is 100-500 times slower than lipids.  Most membrane protein diffusion is limited by aggregation or attachment to cytoskeleton.  Can examine distribution of membrane proteins by freeze-fracture electron microscopy.  Membrane fluidity is dependent upon the flexibility of fatty acyl chains.  Fully extended saturated fatty acyl chains show maximum van der Waals interactions.  When heated, the chains become disordered --> less interactions --> membrane “shrinks” in size due to less extension of tails --> due to rotation around C-C bond.  For lipids with unsaturated acyl chains, kink disrupts ordered packing and increases membrane fluidity --> decreases phase transition temperature (becomes more fluid at lower temperature).  Some organisms can alter their membrane fluidity by adjusting the ratio of unsaturated to saturated fatty acids. e.g. bacteria grown at low temperature increase the proportion of unsaturated fatty acyl groups. e.g. warm-blooded animals have less variability in that ratio because of the lack of temperature fluctuations. exception: reindeer leg has increased number of fatty acyl groups as get closer to hoof --> membrane can remain more fluid at lower temperatures.  Cholesterol also affects membrane fluidity.  Accounts for 20-25% of lipid mass of membrane.  Broadens the phase-transition temperature.  Intercalation of cholesterol between membrane lipids restricts mobility of fatty acyl chains ---> fluidity decreases.  Helps maintain constant membrane fluidity despite changes in temperature and degree of fatty acid saturation.

CHAPTER 10 : Lipid Metabolism

Fatty acids have four major physiologic roles in the cell:  Building blocks of phospholipids and glycolipids  Added onto proteins to create lipoproteins, which targets them to membrane locations  Fuel molecules - source of ATP  Fatty acid derivatives serve as hormones and intracellular messengers 91 STUDY GUIDE for BIOCHEMISTRY 2009

Absorption and Mobilization of Fatty Acids  Most lipids are triacylglycerols, some are phospholipids and cholesterol.  Digestion occurs primarily in the small intestine.  Fat particles are coated with bile salts (amphipathic) from gall bladder.  Degraded by pancreatic lipase (hydrolyzes C-1 and C-3 ---> 2 fatty acids and 2-monoacylglycerol).  Can then be absorbed by intestinal epithelial cells; bile salts are recirculated after being absorbed by the intestinal epithelial cells.  In the cells, fatty acids are converted by fatty acyl CoA molecules.  Phospholipids are hydrolyzed by pancreatic phospholipases, primarily phospholipase A . 2  Cholesterol esters are hydrolyzed by esterases to form free cholesterol, which is solubilized by bile salts and absorbed by the cells.  Lipids are transported throughout the body as lipoproteins.  Lipoproteins consist of a lipid (tryacylglycerol, cholesterol, cholesterol ester) core with amphipathic molecules forming layer on outside.

Lipoproteins

 Both transported in form of lipoprotein particles, which solubilize hydrophobic lipids and contain cell-targeting signals.  Lipoproteins classified according to their densities: o chylomicrons - contain dietary triacylglycerols o chylomicron remnants - contain dietary cholesterol esters o very low density lipoproteins (VLDLs) - transport endogenous triacylglycerols, which are hydrolyzed by lipoprotein lipase at capillary surface o intermediate-density lipoproteins (IDL) - contain endogenous cholesterol esters, which are taken up by liver cells via receptor- mediated endocytosis and converted to LDLs o low-density lipoproteins (LDL) - contain endogenous cholesterol esters, which are taken up by liver cells via receptor-mediated endocytosis; major carrier of cholesterol in blood; regulates de novo cholesterol synthesis at level of target cell o high-density lipoproteins - contain endogenous cholesterol esters released from dying cells and membranes undergoing turnover Storage of Fatty Acids  Triacylglycerols are transported as chylomicrons and VLDLs to adipose tissue; there, they are hydrolyzed to fatty acids, which enter adipocytes and are esterified for storage.  Mobilization is controlled by hormones, particularly epinephrine, which binds to  -adrenergic receptors on adipocyte membrane --> protein kinase

92 STUDY GUIDE for BIOCHEMISTRY 2009 A activated --> phosphorylates hormone-sensitive lipase --> converts triacylglycerols to free fatty acids and monoacylglycerols.  Insulin inhibits lipid mobilization (example of reciprocal regulation).  Monoacylglycerols formed are phosphorylated and oxidized to DHAP (intermediate of glycolysis and gluconeogenesis).

ATP ADP NAD+ NADH + H+

glycerol glycerol 3-phosphate dihydroxyacetone phosphate glycerol kinase glycerol phosphate dehydrogenase

Can be converted to glucose (gluconeogenesis) or pyruvate (glycolysis) in the liver.

Fatty Acid Oxidation ( -oxidation)  Fatty acids are degraded by oxidation of the  carbon by-oxidation.  Pathway that removes 2-C units at a time --> acetyl CoA --> citric acid cycle --> ATP  There are three stages in  -oxidation: o Activation of fatty acids in cytosol catalyzed by acyl CoA synthetase; two high energy bonds are broken to produce AMP o 2) Transport of fatty acyl CoA into mitochondria via carnitine shuttle o 3)  -oxidation - cyclic pathway in which many of the same enzymes are used repeatedly (see pathway sheet)

-oxidation of odd chain and unsaturated fatty acids

 Odd chain fatty acids undergo  -oxidation until propionyl CoA is formed.  Propionyl CoA is then converted to succinyl CoA, which then enters the Krebs cycle.  See pathway sheet for details

 Unsaturated fatty acids need two additional enzymes besides those of - oxidation. o enoyl-CoA isomerase o 2,4-dienoyl-CoA reductase  How the pathway looks depends upon the location of the double bond, but there are two possibilities.  See pathway sheets for details.

ATP generation from Fatty Acid Oxidation:

 Can be estimated from the amount of acetyl CoA, QH2, and NADH produced.  See pathway sheet.

Regulation of Fatty Acid Oxidation

 Already talked about fatty acid mobilization via epinephrine.  Net result is high concentrations of acetyl CoA and NADH via  -oxidation.  Both molecules allosterically inhibit pyruvate dehydrogenase complex.  Most of acetyl CoA produced goes to Krebs cycle; during periods of fasting, excess acetyl CoA is produced, too much for Krebs cycle.  Also in diabetes, oxaloacetate is used to form glucose by gluconeogenesis --> concentration of oxaloacetate is lowered.  Result is the diversion of acetyl CoA to form acetoacetate and 3- hydroxybutyrate; these two molecules plus acetone are known as ketone bodies.  Acetoacetate is formed via the following reactions:

acetyl CoA CoA acetyl CoA 2 acetyl CoA 3-hydroxy- acetoacetate HMG-CoA lyase 3-methylglutaryl CoA

NADH + H+ -hydroxy H+ + NAD butyrate CO2 Dehydrogenase

3-hydroxybutyrate acetone

 The major site of ketone body synthesis is the liver, within the mitochondrial matrix ---> transported to the bloodstream.  Acetoacetate and 3-hydroxybutyrate are used in respiration and are important sources of energy.  Cardiac muscle and the renal cortex perferentially use acetoacetate over glucose.  Glucose is used by brain and RBCs; in brain, ketone bodies substitute for glucose as fuel because the brain cannot undergo gluconeogenesis.  Acetoacetate can be converted to acetyl CoA and oxidized in citric acid cycle only in nonhepatic tissues.

Diabetes (insulin-dependent diabetes mellitus; IDDM) Decreased insulin secretion by beta cells of pancreas; could be caused by viruses (?) 94 STUDY GUIDE for BIOCHEMISTRY 2009 Juvenile onset Patients are thin, hyperglycemic, dehydrated, polyuric (pee a lot), hungry, thirsty In these patients, glycogen mobilization, gluconeogenesis, fatty acid oxidation occurs --- > massive ketone body production; also, some of the glucose is in urine (tends to pull water out of body) ----> diabetic ketoacidosis

FATTY ACID SYNTHESIS

Important features of this pathway: 1) Synthesis takes place in cytosol;  -oxidation takes place in mitochondrial matrix. 2) Intermediates are bound to sulfhydral groups of acyl carrier protein (ACP); intermediates of -oxidation are bonded to CoA 3) Growing fatty acid chain is elongated by sequential addition of two-carbon units derived from acetyl CoA

4) Reducing power comes from NADPH; oxidants in  -oxidation are NAD+ and FAD 5) Elongation of fatty acid stops when palmitate (C16) is formed; further elongation and insertion of double bonds carried out later by other enzymes

Fatty acid synthesis takes place in three stages: 1) Mitochondrial acetyl CoA is transported into cytosol via citrate transport system Acetyl CoA is condensed with oxaloacetate to form citrate ---> antiported out with inward movement of anion Citrate cleaved by cytosolic citrate lyase --> oxaloacetate + acetyl CoA 2) Formation of malonyl CoA Acetyl CoA carboxylase is key regulatory enzyme Influenced by glucagon --> inactivates enzyme in liver Epinephrine inactivates enzyme in adipocytes Citrate allosterically activates enzyme Fatty acyl CoA allosterically inhibits enzyme 3) Assembly of fatty acid chain via fatty acid synthase Consists of five separate stages: 1) Loading - acetyl CoA and malonyl CoA are attached to acyl carrier protein 2) Condensation - both are condensed by fatty acid synthase to from acetoacetyl-ACP 3) Reduction - NADPH is oxidized to form hydroxybutyryl ACP 4) Dehydration - formation of double bond 5) Reduction - NADPH is source of e- and H+ to form butyrylACP

Last four steps are repeated, each time with malonyl-ACP to elongate chain, until palmitate is produced.

Overall reaction:

+ acetyl CoA + 7 malonyl CoA + 14 NADPH + 20 H ---> palmitate + 7CO2 + 14 + NADP + 8 HS-CoA + 6 H2O

Regulation of Fatty Acid Synthesis

 Metabolism of fatty acids is under hormonal regulation by glucagons, epinephrine, and insulin.  Fatty acid synthesis is maximal when carbohydrate and energy are plentiful.  Important points of control are release of fatty acids from adipocytes and regulation of carnitine acyltransferase I in the liver.  High insulin levels also stimulate formation of malonyl CoA, which allosterically inhibits carnitine acyltransferase I  fatty acids remain in cytosol and are not transported to mitochondria for oxidation.  Key regulatory enzyme is acetyl-CoA carboxylase (catalyzes first committed step in fatty acid synthesis).  Insulin stimulates fatty acid synthesis and inhibits hydrolysis of stored triacylglycerols.  Glucagon and epinephrine inhibit fatty acid synthesis (enzyme is phosphorylated by protein kinase A; removal of phosphate group catalyzed by protein phosphatase 2A).  Citrate is an allosteric activator, but its biological relevance has not been established.  Fatty acyl CoA acts as an inhibitor.  Palmitoyl CoA and AMP are allosteric inhibitors.

Synthesis of Eicosanoids

 Precursors for eicosanoids are 20-carbon polyunsaturated fatty acids such as arachidonate.  Part of inner leaflet of cell membrane.  There are two classes of eicosanoids: 1) prostaglandins and thromboxanes Synthesized by enzyme cyclooxygenase Localized molecules such as thromboxane A2, prostaglandins, prostacyclin ae produced. Thromboxane A2 leads to platelet aggregation and blood clots  reduced blood flow in tissues. Aspirin binds irreversibly to COX enzymes and prevents prostaglandin synthesis. 2) leukotrienes Produced by lipoxygenases. 96 STUDY GUIDE for BIOCHEMISTRY 2009 Products were once called “slow-acting substances of anaphylaxis”, responsible for fatal effects of some immunizations.

Synthesis of Triacylglycerols and Glycerophospholipids

Most fatty acids are esterified as triacylglycerols or glycerophospholipids. Intermediate molecule in synthesis of these two molecules is phosphatidic acid or phosphatidate.

There are two pathways: 1) de novo – “from scratch” 2) salvage pathway - uses “old” pieces and parts to make new molecules

Synthesis of phosphatidate:  Common intermediate in synthesis of phosphoglycerides and triacylglycerols  Formed from glycerol 3-phosphate and 2 acetyl CoA molecules  Enzyme is glycerol phosphate acyltransferase

Synthesis of triacylglycerols and neutral phospholipids:  Uses phosphatidate, which is dephosphorylated to produce 1,2- diacylglycerol If acetylated ---> triacylglyerol If reacted with nucleotide derivative --> phosphatidylcholine or phosphatidylethanolamine

Synthesis of acidic phospholipids:  Uses phosphatidate and reacts it with CTP ---> CDP-diacylglycerol  Addition of serine --> phosphatidylserine  Addition of inositol ---> phosphatidylinositol  In mammals, phosphatidylserine and phosphatidylethanolamine can be interconverted - base-exchange occurs in ER.  Decarboxylation occurs in mitochondria and procaryotes

Synthesis of Sphingolipids

 All have C unsaturated alcohol (sphingosine) as structural backbone, 18 rather than glycerol  Palmitoyl CoA and serine condense ---> dehydrosphinganine ---> sphingosine  Acetylation of amino group of sphingosine ---> ceramide  Substitution of terminal hydroxyl group gives: 97 STUDY GUIDE for BIOCHEMISTRY 2009  sphingomyelin -- addition of phosphatidylcholine  cerebroside -- substitute UDP-glucose or UDP-galactose  gangliosides -- substitute oligosaccharide

Tay-Sachs disease = inherited disorder of ganglioside breakdown.  Deficient or missing enzyme is  -N-acetylhexosaminidase, which removes the terminal N-acetylgalactosamine residue from its ganglioside.  One in 30 Jewish Americans of eastern European descent are carriers of a defective allele.  Can be diagnosed during fetal development by assaying amniotic fluid for enzyme activity.  Causes weakness, retarded psychomotor development, blindness by age two, and death around age three.

Synthesis of Cholesterol

 Precursor of steroid hormones and bile salts.  Most cholesterol is synthesized in liver cells, although most animal cells can synthesize it.  Starts with 3 molecules of acetyl CoA to form 3-hydroxy-3-methyl-glutaryl CoA, which is reduced to mevalonate (C6) by HMG-CoA reductase (first committed step of cholesterol synthesis)  Amount of cholesterol formation by liver and intestine is highly responsive to cellular levels of cholesterol.  Enzyme HMG-CoA reductase is controlled in multiple ways: 1) Rate of enzyme synthesis is controlled by sterol regulatory element (SRE); SRE inhibits mRNA production 2) Translation of reductase mRNA is inhibited by nonsterol metabolites derived from mevalonate 3) Degradation of the enzyme occurs at high enzyme levels 4) Phosphorylation of enzyme

If enzyme is phosphorylated via glucagon pathway --> decreased activity-->

cholesterol synthesis ceases when ATP levels are low

If enzyme is dephosphorylated via insulin pathway --> increased activity

 Cells outside liver and intestine obtain cholesterol from blood instead of synthesizing it de novo.  Steps in the uptake of cholesterol by LDL pathway: 1) apolipoprotein on surface of LDL particle binds to receptor on membrane of nonhepatic cells 98 STUDY GUIDE for BIOCHEMISTRY 2009 2) LDL-receptor complex internalized by endocytosis 3) vesicles formed fuse with lysosomes, which breaks apart protein part of lipoprotein to amino acids and hydrolyzes cholesterol esters 4) released unesterified cholesterol can be used for membrane biosynthesis or be reesterified for storage

 Defects in LDL receptor lead to familial hypercholesterolemia (FH), in which cholesterol and LDL levels are markedly elevated.  Result is deposition of cholesterol in tissues because of high levels of LDL- cholesterol in blood  Heterozygotes suffer from atherosclerosis and increased risk of stroke  Homozygotes usually die in childhood from coronary artery disease  Disease is the result of an absence (homozygotes) or reduction (heterozygotes) in number of LDL receptors.  LDL entry into liver and other cells is impaired.  Drug therapy can help heterozygotes 1) can inhibit intestinal absorption of bile salts (which promote absorption of dietary cholesterol) 2) lovastatin - competitive inhibitor of HMG-CoA reductase ---> blocks cholesterol synthesis

CHAPTER 11 : Nucleotides, nucleic acids Nucleic Acids Form Complementary Interactions

How is the potential for copying primary sequency made possible in nucleic acids? The structure of nucleic acid makes it possible to form a duplex of two complementary chains The chains are complementary because each nucleic acid residue can form a unique complementary interaction with another residue In DNA, there are 4 types of residues (A, G, C, T) which form two complementary interactions (A-T, G-C):

Complementary DNA Chains Form a Helical Structure Two DNA chains that have a complementary sequence can form a doublestranded helix:

100 STUDY GUIDE for BIOCHEMISTRY 2009 Each of the two chains serves as a template for the formation of a new complementary chain

As the original helix separates and new complementary chains are created, the result is a copy of the original chain:

The Flow of Genetic Information This ability to copy the information in a DNA molecule enables a flow of information in the cell The stable information within DNA is copied to temporary messages of RNA which in turn are used to create Protein:

A second flow of information occurs between cells, from one generation to the next

Nucleotides

Each residue in a nucleic acid is composed of three components: a ribose sugar a base a phosphate group

The Ribose Component

The ribose is a 5-membered ring The atoms in the ring are numbered with a prime (') symbol, to distinguish them from the numbered atoms in the base. Note that the sugars in DNA residues lack an oxygen on the 2' carbon, so it is called deoxyribose

The Base Component The second component of a nucleic acid residue is a planar, aromatic base There are two kinds of bases: purines and pyrimidines

The Phosphate Group

The third component is the phosphate group By convention, the phosphate group of a residue is the one connected to the 5' carbon of the ribose At physiological pH, the phosphate group of each residue has a single negative charge, which results in a large overall negative charge on the entire nucleic acid chain In solution, the negative charges on the phosphates are compensated by nearby positively-charged counter-ions or positively-charged side chains of nucleic-acid

103 STUDY GUIDE for BIOCHEMISTRY 2009 binding proteins (lysines and arginines)

Nucleosides

The structure of the ribose and the base together, without the phosphate, is referred to as a nucleoside

The four nucleosides in RNA are adenosine guanosine cytidine uridine The four nucleosides in DNA are deoxyadenosine deoxyguanosine dexoycytidine deoxythymidine (thymidine)

Nucleoside Structure

In nuclosides, the base is attached to the ribose at the C1' carbon in a - glycosidic linkage:

104 STUDY GUIDE for BIOCHEMISTRY 2009 This is the bond from the anomeric carbon that comes up out of the plane of a sugar

In nucleosides with purine bases, the C1' carbon connects to the N9 atom of the purine In nucleosides with pyrimidine bases, the C1'carbon connects to the N1 atom of the pyrimidine Nucleotides

105 STUDY GUIDE for BIOCHEMISTRY 2009 When a nucleoside is joined to one or more phosphates, it is referred to as a nucleotide In DNA, phosphates can be attached at the 5' and the 3' carbons In RNA, phosphates can be attached at the 5', 3' and 2' carbons A nucleoside with a group attached to the 5' end is a nucleoside 5'-phosphate For example, the nucleotide ATP has a triphosphate group at its 5' end, so it is called 5'- ATP or adenosine 5'-triphosphate or adenylate The type of nucleoside and the number and position of the phosphate groups affect the naming of the nucleotide

The directionality of nucleic acid is shown in a 2D structural representation of a tetranucleotide of DNA: pdApdGpdTpdC, or more commonly, just AGTC The individual residues are linked together by 3'-5' phosphodiester covalent bonds The 'backbone' of the structure consists of the phosphoryl groups and the deoxyribose moieties Note the unit negative charge for each residue in the chain, at normal physiological pH

The DNA Helix

As mentioned previously, two nucleic acid chains with a complementary sequence can associate to form a double helix Depending upon the type of nucleic acid and the conditions, two types of helices are commonly found: the A-form helix and the B-form helix Two complementary strands of DNA can adopt either the A-form or the B-form helix, depending upon the conditions One DNA strand and one RNA strand or two RNA strands will associate only in the Aform geometry

The B-Form Double Helix Under normal physiological conditions, DNA is found in the B-form double helix The double helix is a right-handed helix, with the two strands running in antiparallel orientation:

There are 10 residues per turn, with a pitch of 34A The diameter of the helix is 20A

The Helix is Stabilized by Base Pairs

The flat nucleotide bases lie perpendicular to the helix axis and point inward toward the center

Two bases on opposite strands form a complementary interaction called a base pair The base pairing results from hydrogen bonds that form at the base edges Adenine forms two h-bonds with thymine, guanine forms three h-bonds with cytosine

Chemical Structure of Double-Stranded DNA

The 2D structural representation of a doublestranded sequence of DNA shows the two strands running in opposite or antiparallel orientations The negatively-charged phosphate groups are on the outside and the bases are hydrogen bonded in the interior. The negatively-charged phosphate groups are on the outside and the bases are hydrogen bonded in the interior. Note again that in the actual 3D structures, the planes of the bases are oriented mostly perpendicular to the plane of the ribose sugars in the backbone