Chapt. 8 Enzymes as catalysts Ch. 8 Enzymes as catalysts Student Learning Outcomes: Explain general features of enzymes as catalysts: Substrate -> Product Describe nature of catalytic sites general mechanisms Describe how enzymes lower activation energy of reaction Explain how drugs and toxins inhibit enzymes Describe 6 categories of enzymes

Catalytic power of enzymes Enzymes do not invent new reactions Enzymes do not change possibility of reaction to occur (energetics) Enzymes increase the rate of reaction by factor of 1011 or higher Fig. 8.1 box of golfballs, effect of browning enzyme

Enzymes catalyze reactions Enzymes provide speed, specificity and regulatory control to reactions Enzymes are highly specific for biochemical reaction catalyzed (and often particular substrate) Enzymes are usually proteins (also some RNAs = ribozymes) E + S ↔ ES binding substrate ES ↔ EP substrate converted to bound product EP ↔ E + P release of product

Glucokinase is a typical enzyme Glucokinase is typical enzyme: ATP: D-glucose 6-phosphotransferase Very specific for glucose Not phosphorylate other hexoses Only uses ATP, not other NTP 3D shape of enzyme critical for its function (derived from aa sequence) Fig. 8.2 glucokinase

Enzyme active site does catalysis A. Active site of enzyme Enzyme active site does catalysis Substrate binds cleft formed by aa of enzyme Functional groups of enzyme, also cofactors bond to substrate, perform the catalysis; Fig. 8.4

B. Binding site specificity Substrate binding site is highly specific ‘Lock-and-key’ model: 3D shape ‘recognizes substrate (hydrophobic, electrostatic, hydrogen bonds) ‘Induced-fit’ model: enzyme conformational change after binding substrate galactose differs from glucose, needs separate galactokinase Fig. 8.5 glucokinase

Glucokinase conformational change Conformation change of glucokinase on binding glucose Binding positions substrate to promote reactions Large conformational change adjusts actin fold, and facilitates ATP binding Actin fold named for G-actin (where first described; Fig. 7.8) Fig. 8.6 glucokinase (Yeast hexokinase)

Transition state complex Energy Diagram: substrates are activated to react: Activation energy: barrier to spontaneous reaction Enzyme lowers activation energy Transition-state complex is stabilized by diverse interactions Fig. 8.7

Transition-state complex Transition-state complex binds enzyme tightly: transition-state analogs are potent inhibitors of enzymes (more than substrate analogs) make prodrugs that convert to active analogs at site of action Abzymes: catalytic antibodies that have aa in variable region like active site of transition enzyme: Artificial enzymes: catalyze reaction Ex. Abzyme to Cocaine esterase destroys cocaine in body

II. Catalytic mechanism of chymotrypsin - example enzyme Chymotrypsin, serine protease, digestive enzyme: Hydrolyzes peptide bond (no reaction without enzyme) Serine forms covalent intermediate Unstable oxyanion (O-) intermediate Cleaved bond is scissile bond Fig. 8.8

B. Catalytic mechanism of chymotrypsin 1. Specificity of binding: Tyr, Phe, Trp on denatured proteins Oxyanion tetrahedral intermediate His57, Ser195, Asp 2. acyl-enzyme intermediate 3. Hydrolysis of acyl-enzyme intermediate Fig. 8.9

Mechanism of chymotrypsin, cont. 3. Hydrolysis of acyl- enzyme intermediate Released peptide product Restores enzyme Fig. 8.9

Energy diagram revisited with detail Chymotrypsin reaction has several transitions: See several steps Lower energy barrier to uncatalyzed Fig. 8.10

III. Functional groups in catalysis All enzymes stabilize transition state by electrostatic Not all enzymes form covalent intermediates Some enzymes use aa of active site (Table 1): Ser, Lys, His - covalent links His - acid-base catalysis peptide backbone – NH stabilize anion Others use cofactors (nonprotein): Coenzymes (assist, not active on own) Metal ions (Mg2+, Zn2+, Fe2+) Metallocoenzymes (Fe2+-heme)

Coenzymes assist catalysis Activation-transfer coenzymes: Covalent bond to part of substrate; enzyme completes Other part of coenzyme binds to the enzyme Ex. Thiamine pyrophosphate is derived from vitamin thiamine; works with many different enzymes enzB takes H from TPP; carbanion attacks keto substrate, splits CO2 Fig. 8.11

Other activation-transfer coenzymes Specific chemical group binds enzyme Other functional group participates directly in reaction Depends on enzyme for specificity of substrate, catalysis Fig. 8.12 A CoA forms thioesters with many acyl groups: acetyl, succinyl, fatty acids

Oxidation-reduction coenzymes Oxidoreductase enzymes use other coenzymes: Oxidation is loss of electrons (loss H, or gain O) Reduction is gain electrons (gain H, loss of O) Redox coenzymes do not form covalent bond to substrate Unique functional groups NAD+ (and FAD) special role for ATP generation: Ex. Lactate dehydrogenase oxidizes lactate to pyruvate transfers e- & H: to NAD+ -> NADH Fig. 8.13 lactate dehydrogenase

Metal ions assist in catalysis Positive metal ions attract electrons: contribute Mg2+ often bind PO4, ATP; ex. DNA polymerases Some metals bind anionic substrates Fig. 8.14 ADH alcohol dehydrogenase oxidizes alcohol to acetaldehyde and NAD+ to NADH Zn2+ assists with NAD+ (In Lactate dehydrogenase, a His residue assisted the reaction)

pH affects enzyme activity Each enzyme has characteristic pH optimum: Depends on active-site amino acids Depends on H bonds required for 3D structure Each enzyme has optimum temperature for activity: Humans 37oC Taq polymerase for PCR: 72oC Fig. 8.15 optimal pH for enzyme

V. Mechanism-based inhibitors Inhibitors decrease rate of enzyme reaction: Mechanism-based inhibitors mimic or participate in intermediate step of reaction; Covalent inhibitors Transition-state analogs Heavy metals Fig. 8.2 organophosphate inhibitors include two insecticides, and nerve gas Sarin

Covalent inhibitors Covalent inhibitors form covalent or very tight bonds with functional groups in active site: Fig. 8.16 DFP di-isopropylfluorophosphate prevents acetylcholinesterase from degrading acetylcholine

Transition state analogs Transition-state analogs bind more tightly to enzyme than substrate or product: Penicillin inhibits glycopeptidyl transferase, enzyme that synthesizes cross-links in bacterial cell wall. Kills growing cells by inactivating enzyme Fig. 8.17 penicillin

Allopurinol treats gout Allopurinol is suicide inhibitor of xanthine oxidase: Treatment for gout (decreases formation of urate) Fig. 8.18

Basic reactions and classes of enzymes 6 basic classes of enzymes: Oxidoreductases Oxidation-reduction reactions (one gains, one loses e-) Transferases Group transfer – functional group from one to another Hydrolases cleave C-O, C-N and C-S bonds addition of H2O in form of OH- and H+ Lyases diverse cleave C-C, C-O, C-N Isomerases rearrange, create isomers of starting Ligases synthesize C-C, C-S, C-O and C-N bonds; Reactions often use cleavage of ATP or others

Some example enzymes Example enzymes: Group transfer – transamination transfer of amino group Isomerase – rearranges atoms ex. In glycolysis Fig. 8.19

Enzymes are proteins (or RNA) that are catalysts Key concepts Enzymes are proteins (or RNA) that are catalysts accelerate rate of reaction Enzymes are very specific or substrate Enzymes lower energy of activation – to reach high- energy intermediate state Functional groups at active site (amino acid residues, metals, coenzymes) cause catalysis Mechanisms of catalysis include: acid-base, formation covalent intermediates, transition state stabilization

Review questions 4. The reaction shown fits into which classification? Group transfer Isomerization Carbon-carbon bond breaking Carbon-carbon bond formation Oxidation-reduction 5. The type of enzyme that catalyzes this reaction is which of the following? Kinase Dehydrogenase Glycosyltransferase Transaminase isomerase