Lecture 3.2: Enzyme Reaction Mechanisms

Question 1

Features of Catalytic Reactions Mechanisms

Answer 1

• show the optimized microenvironment: substrates bind to active site through noncovalent weak interactions
• illustrate the principle of transition state stabilization
• Give the classic examples of acid-base & covalent catalysis
• informed design of HIV protease and SARs coronavirus protease inhibitors

Question 2

Serine protease mechanisms involves [ ] to cleave peptide bonds

Answer 2

acid-base catalysis
covalent catalysis

Question 3

Serine protease family includes

Answer 3

chymotrypsin
trypsin
elastase

conserved tertiary structure despite limited sequence similarity

Question 4

The active sites of serine proteases share common structural components: What are they?

Answer 4

binding pocket
catalytic triad
oxyanion hole

Question 5

Binding pocket

Answer 5

determine substrate specificity

Question 6

catalytic triad

Answer 6

3 conserved amino acid residues

Question 7

Oxyanion hole

Answer 7

active site close to catalytic triad
negatively charged O2 [transition state during rxn]

Question 8

Pocket Properties?

Chymotrypsin

Answer 8

deep, hydrophobic
made of 2 glycines + serine

Question 9

Pocket properties?

Trypsin

Answer 9

deep pocket, negative charge at bottom
made of 2 glycines + 1 aspartate

Question 10

pocket properties

Elastase

Answer 10

shallow, hydrophobic pocket
made up of 1 threonine + 1 valine + 1 serine

smaller than other 2

Question 11

What amino acids bind to chymotrypsin?

Answer 11

phenylalanine
tyrosine
tyrptophan

aromatic + bulky

Question 12

What amino acids bind to trypsin?

Answer 12

lysine
arginine
NOT histidine because too bulky

Question 13

what amino acids bind to elastase?

Answer 13

glycine
alanine

Question 14

All serine proteases use catalytic triad consisting of three [ ] amino acids: [ ]

Answer 14

conserved
ser, his, asp

Question 15

What two amino acids function together to make Ser into a highly reactive nucleophile?

Answer 15

Asp and His

Question 16

Asp and His Steps to making Ser a highly reactive nucleophile

Answer 16

1. Asp makes His imidazole side chain more basic (a negative charged oxygen stabilizes His and makes more basic His residue)
2. His removes proton from Ser making Ser more nucleophile (His acts as good base catalyst and removes proton from ser anc creates negative charge oxygen)
3. Serine can then act as nucleophile (break covalent peptide bond)

Question 17

The tetrahedral intermediate is stabilized by [ ] with backbone [ ] groups in the [ ]

Answer 17

hydrogen bonds
amide
oxyanion hole

Question 18

Chymotrypsin Reaction Mechanism:

Step 1

Answer 18

• substrate binds to the active site after an interaction between the aromatic side chain and specificity pocket
• polypeptide substrate binds to enzyme active site
• substrate binds to enzyme with high specificity

Question 19

Chymotrypsin Reaction Mechanism:

Step 2

Answer 19

• nucleophilic attack by the Ser oxygen on the carbonyl carbon results in the formation of an acyl-enzyme intermediate
• His57 removes a proton from Ser 195 –> nucleophillic attack by Serine
• Oxyanion is stablized in oxyanion hole made by backbone amino acid groups gly193 and ser195

Question 20

Chymotrypsin Reaction Mechanism:

Step 3

Answer 20

• His57 donates a proton to the amino group of substrate, facilitating peptide bond cleavage
• His 57 donates a proton to the amino group of the substrate, resulting in peptide bond cleavage
• product 1 released

Question 21

Chymotrypsin Reaction Mechanism:

Step 4

Answer 21

• the nucleophilic attack by OH- on the carbonyl carbon leads to the formation of 2nd tetrahedral intermediate
• water enters the active site
• His57 = general base
• oxyanion hole stabilized

Question 22

Chymotrypsin Reaction Mechanism:

Step 5 & 6

Answer 22

• His57 donates a proton to Ser195 resulting in cleavage of acyl-enzyme intermediate and regeneration of the catalytic triad
• Amino-terminal fragment is released as the second product

Question 23

Key Points of Chymotrypsin Reaction Mechanism:

positioning of catalytic triad amino acids in the active site creates the [ ] that can cleave the peptide bond

Answer 23

nucleophile

Question 24

Key Points of Chymotrypsin Reaction Mechanism:

positioning of Gly193 and Ser195 form a [ ] which stabilizes [ ]

Answer 24

microenvironent (oxyanion hole)
an otherwise unstable tetrahedral intermediate

Question 25

Key points of Chymotrypsin Reaction Mechanism:

His serves as [ ] catalytst where it withdraws a proton to increase the [ ] of [ ] and donates a proton to resolve the [ ] tetrahedral intermediate

Answer 25

general acid-base
nucleophilicity of Ser
2nd tetrahedral intermediate

Question 26

HIV Protease

Uses which two types of catalysis

Answer 26

acid-base
covalent catalysis

Question 27

Enolase Reaction Mechanisms

Uses which two types of catalysis to do what?

Answer 27

general acid-base catalysis and metal ion catalysis to eliminate a water molecule

Question 28

Mg2+ Metal Ion Catalysis

Mg2+ ions orient the [ ] in the [ ] and stabilized the [ ] intermediate

Answer 28

substrate
active site
carbanion