Enzyme mechanisms: Chymotrypsin, Lysozyme and HIV protease Flashcards Preview

BIOC 4331 > Enzyme mechanisms: Chymotrypsin, Lysozyme and HIV protease > Flashcards

Flashcards in Enzyme mechanisms: Chymotrypsin, Lysozyme and HIV protease Deck (14)
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1
Q

Protease

A

Proteases catalyze the hydrolysis of polypeptide backbones to yield smaller peptides

2
Q

What/where does trypsin cleave?

A

cleaves C terminal side of R,K

3
Q

What/where does chymotrypsin cleave?

A

cleaves C terminal side of F,Y,W

4
Q

What/where does elastase cleave?

A

cleaves smaller, non-polar amino acids A,G,S,V

5
Q

What is the catalytic triad of chymotrypsin?

A

Ser195 and His57 plus a third invariant residue in serine proteases is Asp102, which is buried in a solvent-inaccessible pocket.

6
Q

What is the first step in the serine protease mechanism?

A
  1. Binding and formation of ES complex
7
Q

What gives serine protease specificity to its substrate?

A

There is a hydrophobic pocket in the active site that conforms to the aromatic ring in the peptide to be cleaved

8
Q

What is the second step in the serine protease mechanism?

A
  1. Nucleophilic attack by Ser OH.

A. His57 deprotonates Ser195 OH – general base catalysis

B. Ser O- attacks carbonyl carbon (nucleophilic) – proximity and orientation

C. Forming tetrahedral intermediate 1 – covalent catalysis

9
Q

What is the third step in the serine proetase mechanism?

A
  1. Protonation of peptide amino group.

His 57 protonates peptide amino group – general acid catalysis

Peptide bond is cleaved

N-terminal fragment still attached to Ser 195 (acyl- enzyme intermediate)

Neutron diffraction shows that the proton between His57 and Asp102 remains with His (electrostatic catalysis)

10
Q

What is the fourth step in the serine protease mechanism?

A
  1. Deprotonation of H2O and attack carbonyl carbon (H2O is second substrate)

A. His57 acts as a base to abstract proton from H2O – general base catalysis.

(note: pH must be high enough so that His57 isn’t protonated!)

B. Resulting hydroxyl ion attacks carbonyl forming tetrahedral intermediate 2.

11
Q

What effect does pH have on the serine protease mechanism?

A

pH must be high enough so that His57 isn’t protonated!

Step 4 of the mechanism requires His57 to be deprotonated so that it can abstract a proton from the aqueous solute so that hydroxyl ion can attack the substrate and form tetrahedral intermediate 2.

12
Q

Which intermediate in the serine protease mechanism is the least stable?

A

Intermediate 2

It is stabilised by the oxianion hole which accomidates the oxyanion in tetrahedral intermediate 2

Peptide N’s form good hydrogen bonds with oxyanion, but not when carbonyl is in a trigonal conformation, e.g. ES complex or acyl-enzyme intermediate

Thus the oxyanion hole enables preferential binding to the transition state

13
Q

oxyanion hole in serine protease

A

The oxianion hole accomidates the oxyanion in tetrahedral intermediate 2 and stabilses it

Peptide N’s form good hydrogen bonds with oxyanion, but not when carbonyl is in a trigonal conformation, e.g. ES complex or acyl-enzyme intermediate

Thus the oxyanion hole enables preferential binding to the transition state

14
Q

Explain the catalytic rate of serine protease in relation to pH

A

chymotrypsin His57 pKa ~ 7