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Chapt. 8 Enzymes as catalysts Catalytic power of enzymes Ch. 8 Enzymes as catalysts Enzymes do not invent new reactions Student Learning Outcomes: • Explain general features of enzymes as catalysts: Substrate -> Product • Describe nature of catalytic sites Enzymes do not change possibility of reaction to occur (energetics) • general mechanisms • Describe how enzymes lower activation energy of reaction • Explain how drugs and toxins inhibit enzymes • Describe 6 categories of enzymes 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 • ES ↔ EP binding substrate substrate converted to bound product • EP ↔ E + P release of product Enzymes increase the rate of reaction by factor of 1011 or higher Fig. 8.1 box of golfballs, effect of browning enzyme 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 1 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; 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 Fig. 8.4 Glucokinase conformational change Transition state complex Conformation change of glucokinase on binding glucose Energy Diagram: substrates are activated to react: Activation energy: barrier to spontaneous reaction Enzyme lowers activation energy • Binding positions substrate to promote reactions Transition-state complex is stabilized by diverse interactions • 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) Fig. 8.7 2 Transition-state complex II. Catalytic mechanism of chymotrypsin - example enzyme 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 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 • Abzymes: catalytic antibodies that have aa in variable region like active site of transition enzyme: • • Fig. 8.8 Artificial enzymes: catalyze reaction Ex. Abzyme to Cocaine esterase destroys cocaine in body B. Catalytic mechanism of chymotrypsin Mechanism of chymotrypsin, cont. 1. Specificity of binding: 3. Hydrolysis of acylenzyme intermediate Tyr, Phe, Trp on denatured proteins • Released peptide product Oxyanion tetrahedral intermediate His57, Ser195, Asp • Restores enzyme 2. acyl-enzyme intermediate 3. Hydrolysis of acyl-enzyme intermediate Fig. 8.9 Fig. 8.9 3 Energy diagram revisited with detail Chymotrypsin reaction has several transitions: • See several steps • Lower energy barrier to uncatalyzed III. Functional groups in catalysis 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) Fig. 8.10 Coenzymes assist catalysis Other activation-transfer coenzymes Activation-transfer coenzymes: Activation-transfer coenzymes: • Covalent bond to part of substrate; enzyme completes • Specific chemical group binds enzyme • Other functional group participates directly in reaction • Depends on enzyme for specificity of substrate, catalysis • 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. 8.12 A CoA forms thioesters with many acyl groups: acetyl, succinyl, fatty acids Fig. 8.11 4 Oxidation-reduction coenzymes Metal ions assist in catalysis 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 Positive metal ions attract electrons: contribute • Mg2+ often bind PO4, ATP; ex. DNA polymerases • Some metals bind anionic substrates Fig. 8.14 NAD+ (and FAD) special role for ATP generation: ADH alcohol dehydrogenase • oxidizes alcohol to acetaldehyde and NAD+ to NADH • Zn2+ assists with NAD+ Ex. Lactate dehydrogenase • oxidizes lactate to pyruvate • transfers e- & H: to NAD+ -> NADH (In Lactate dehydrogenase, a His residue assisted the reaction) Fig. 8.13 lactate dehydrogenase pH affects enzyme activity V. Mechanism-based inhibitors 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 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 Fig. 8.15 optimal pH for enzyme 5 Covalent inhibitors Covalent inhibitors form covalent or very tight bonds with functional groups in active site: 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. 8.16 DFP di-isopropylfluorophosphate prevents acetylcholinesterase from degrading acetylcholine Allopurinol treats gout Allopurinol is suicide inhibitor of xanthine oxidase: • Treatment for gout (decreases formation of urate) Fig. 8.17 penicillin 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; Fig. 8.18 • Reactions often use cleavage of ATP or others 6 Some example enzymes Example enzymes: Group transfer – transamination transfer of amino group Isomerase – rearranges atoms ex. In glycolysis Fig. 8.19 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 highenergy 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? a. Group transfer b. Isomerization c. Carbon-carbon bond breaking d. Carbon-carbon bond formation e. Oxidation-reduction 5. The type of enzyme that catalyzes this reaction is which of the following? a. Kinase b. Dehydrogenase c. Glycosyltransferase d. Transaminase e. isomerase 7