1Chapt. 8 Enzymes as catalystsCh. 8 Enzymes as catalystsStudent 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 enzymesCatalytic power of enzymesEnzymes do not invent new reactionsEnzymes do not change possibility of reaction to occur (energetics)Enzymes increase the rate of reaction by factor of 1011or higherFig. 8.1 box of golfballs, effect of browning enzymeEnzymes 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 productGlucokinase is a typical enzymeFig. 8.2 glucokinaseGlucokinase 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)2A. 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 specificityFig. 8.5 glucokinaseSubstrate 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 separategalactokinaseGlucokinase conformational changeFig. 8.6 glucokinase(Yeast hexokinase)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)Transition state complexEnergy Diagram: substrates are activated to react:Activation energy: barrier to spontaneous reactionEnzyme lowers activation energyTransition-state complex is stabilized by diverse interactionsFig. 8.73Transition-state complexTransition-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 bodyII. Catalytic mechanism of chymotrypsin - example enzymeChymotrypsin, serine protease, digestive enzyme:• Hydrolyzes peptide bond (no reaction without enzyme)• Serine forms covalent intermediate• Unstable oxyanion (O-) intermediate• Cleaved bond is scissile bondFig. 8.8B. Catalytic mechanism of chymotrypsin Fig. 8.91. Specificity of binding: Tyr, Phe, Trp on denatured proteinsOxyanion tetrahedral intermediateHis57, Ser195, Asp2. acyl-enzyme intermediate3. Hydrolysis of acyl-enzyme intermediateMechanism of chymotrypsin, cont.Fig. 8.93. Hydrolysis of acyl-enzyme intermediate• Released peptide product• Restores enzyme4Energy diagram revisited with detailChymotrypsin reaction has several transitions:• See several steps• Lower energy barrier to uncatalyzedFig. 8.10 III. Functional groups in catalysisFunctional 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 catalysisFig. 8.11Activation-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 CO2Other activation-transfer coenzymesActivation-transfer coenzymes:• Specific chemical group binds enzyme• Other functional group participates directly in reaction• Depends on enzyme for specificity of substrate, catalysisFig. 8.12 ACoA forms thioesters with many acyl groups:acetyl, succinyl, fatty acids5Oxidation-reduction coenzymesFig. 8.13 lactate dehydrogenaseOxidoreductase 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 groupsNAD+(and FAD) specialrole for ATP generation:Ex. Lactate dehydrogenase• oxidizes lactate to pyruvate• transfers e- & H: to NAD+-> NADHMetal ions assist in catalysisPositive metal ions attract electrons: contribute• Mg2+ often bind PO4, ATP; ex. DNA polymerases• Some metals bind anionic substratesFig. 8.14 ADH alcohol dehydrogenase• oxidizes alcohol to acetaldehydeand NAD+ to NADH• Zn2+ assists with NAD+(In Lactate dehydrogenase, a His residue assisted the reaction)pH affects enzyme activityFig. 8.15 optimal pH for enzymeEach enzyme has characteristic pH optimum:• Depends on active-site amino acids• Depends on H bonds required for 3D structure• Each enzyme has optimumtemperature for activity:Humans 37oCTaq polymerase for PCR: 72oC V. Mechanism-based inhibitorsInhibitors decrease rate of enzyme reaction:• Mechanism-based inhibitors mimic or participate in intermediate step of reaction;• Covalent inhibitors• Transition-state analogs• Heavy metalsFig. 8.2 organophosphate inhibitors include two insecticides, and nerve gas Sarin6Covalent inhibitorsCovalent 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 analogsTransition-state analogs bind more tightly to enzyme than substrate or product:• Penicillin inhibits glycopeptidyl transferase, enzyme that synthesizes cross-links in bacterial