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

Fig. 8.2 glucokinase

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)

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

Fig. 8.5 glucokinase

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 separategalactokinase

Glucokinase conformational change

Fig. 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 complex

Energy Diagram: substrates are activated to react:Activation energy: barrier to spontaneous reactionEnzyme lowers activation energyTransition-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

Fig. 8.9

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

Mechanism of chymotrypsin, cont.

Fig. 8.9

3. Hydrolysis of acyl-enzyme intermediate

• Released peptide product

• Restores enzyme

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

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

Fig. 8.11

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

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. 8.12 ACoA forms thioesters with many acyl groups:acetyl, succinyl, fatty acids

Oxidation-reduction coenzymes

Fig. 8.13 lactate dehydrogenase

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) specialrole 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• 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

Fig. 8.15 optimal pH for enzyme

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 37oCTaq polymerase for PCR: 72oC

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

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?a.Group transferb.Isomerizationc.Carbon-carbon bond breakingd.Carbon-carbon bond formatione.Oxidation-reduction

5. The type of enzyme that catalyzes this reaction is which of the following?a.Kinaseb.Dehydrogenasec.Glycosyltransferased.Transaminasee.isomerase