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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 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)

8. 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

9. 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

10. 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

11. 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

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

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

14. 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)

15. 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

16. 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 A
CoA forms thioesters with many acyl groups:
acetyl, succinyl, fatty acids

17. 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 lactate dehydrogenase

18. 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)

19. 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 optimal pH for enzyme

20. 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

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

22. 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 penicillin

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

24. 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

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

26. 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

27. 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