6.4 Examples of Enzymatic Reactions

Question 1

Protease and example

Answer 1

Enzyme that catalyzes hydrolytic cleavage of peptide bonds. An exaplme is the bovine pancreatic chymotrypsin.

Question 2

Phases and General Mechanism of Chymotrypsin

Answer 2

Acylation phase: cleavage of peptide bond and formation of ester bond between peptide carbonyl carbon and enzyme.
Deacylation phase: hydrolysis of ester linkage and regeneration of nonacylated enzyme.

Question 3

Steps of Chymotrypsin Mechanism

Answer 3

1: Side chain of peptide settles in hydrophobic pocket and positions peptide bond for attack.
2: A ser-his-interaction generates a nucleophilic alkoxide on ser. This ion attacks the carbonyl group of the peptide and forms a tetrahedral acyl-enzyme. This gives the carbonyl oxygen a negative charge, but is stabilized by hydrogen bonding in the oxyanion hole.
3: This negative charge is unstable and collapses the tetrahedral intermediate and reforms the double bond to carbonyl carbon. This process displaces the bond between carbon and the amino group, and the amino group leaves, protonated by His.
4: Incoming water molecule is deprotonated and attacks the hydroxid in the ester bond. A second tetrahedral intermediate is generated with oxygen in the oxyanion hole.
5: The tetrahedral intermediate collapses (for the second time) and results in a carboxylate anion (second product). Ser is displaced.
6: Continuation of the collapse of the tetrahedral intermediate?
7: The second product dissociates from the active site and the free enzyme is regenerated.

Question 4

Important Features of the Chymotrypsin Active Site

Answer 4

Catalytic triad: His, Ser, Asp
Hydrophobic pocket
Oxyanion hole

Question 5

Examples of Proteases

Answer 5

Chymotrypsin, HIV protease

Question 6

Mechanism of HIV Protease

Answer 6

Active site contains two Asp residues. Water molecule in the active site attacks carbonyl carbon and generates a tetrahedral intermediate, stabilized by hydrogen bonds. Collapse of the tetrahedral intermediate leads to a protonated amino acid leaving group.

Question 7

Hexikinase

Answer 7

Undergoes induced fit on substrate binding. Binding of glucose and Mg-ATP induces conformational change to active form. Xylose also induces active conformation, and leads to phosphorylation of water. The mechanisms of the reaction involves acid-base catalysis and transition-state stabilization by active site amino acids.