How To Understand The Chemistry Of An Enzyme

Transcription

1 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 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 ES EP binding substrate 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 1

2 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. 8.4 Fig. 8.5 glucokinase Glucokinase conformational change Conformation change of glucokinase on binding glucose Binding positions substrate to promote reactions 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 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

3 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 Unstable oxyanion (O-) 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 His57, Ser195, Asp 2. acyl-enzyme 3. Hydrolysis of acyl-enzyme Fig. 8.9 Mechanism of chymotrypsin, cont. 3. Hydrolysis of acylenzyme Released peptide product Restores enzyme Fig

4 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 s Some enzymes use aa of active site (Table 1): Ser, Lys, His - covalent links His - acid-base catalysis peptide backbone NH stabilize anion Fig Others use cofactors (nonprotein): Coenzymes (assist, not active on own) Metal ions (Mg 2+, Zn 2+, Fe 2+) Metallocoenzymes (Fe 2+- 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 CO 2 Fig Other activation-transfer coenzymes Activation-transfer coenzymes: Specific chemical group binds enzyme Other functional group participates directly in reaction Depends on enzyme for specificity of substrate, catalysis Fig A CoA forms thioesters with many acyl groups: acetyl, succinyl, fatty acids 4

5 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 Metal ions assist in catalysis Positive metal ions attract electrons: contribute Mg 2+ often bind PO 4, ATP; ex. DNA polymerases Some metals bind anionic substrates Fig ADH alcohol dehydrogenase oxidizes alcohol to acetaldehyde and NAD + to NADH Zn 2+ assists with NAD+ (In Lactate dehydrogenase, a His residue assisted the reaction) Fig lactate dehydrogenase 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 37 o C Taq polymerase for PCR: 72 o C V. Mechanism-based inhibitors Inhibitors decrease rate of enzyme reaction: Mechanism-based inhibitors mimic or participate in step of reaction; Covalent inhibitors Transition-state analogs Heavy metals Fig optimal ph for enzyme Fig. 8.2 organophosphate inhibitors include two insecticides, and nerve gas Sarin 5

6 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 DFP di-isopropylfluorophosphate prevents acetylcholinesterase from degrading acetylcholine Fig penicillin Allopurinol treats gout Allopurinol is suicide inhibitor of xanthine oxidase: Treatment for gout (decreases formation of urate) Fig 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 H 2 O 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 6

7 Some example enzymes Example enzymes: Group transfer transamination transfer of amino group Isomerase rearranges atoms ex. In glycolysis Fig 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 state Functional groups at active site (amino acid residues, metals, coenzymes) cause catalysis Mechanisms of catalysis include: acid-base, formation covalent s, 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