Serine Proteases

serine (Ser, S)
H H3N+ C CH2 COO

Serine proteases include digestive

enzymes trypsin, chymotrypsin, & elastase.

OH

Different serine proteases differ in substrate specificity. For example:

Chymotrypsin prefers an aromatic side chain on the residue whose carbonyl carbon is part of the peptide bond to be cleaved.
Trypsin prefers a positively charged Lys or Arg residue at this position.

Serine Protease Mechanism

A mixture of covalent and general acid-base catalysis Asp-102 functions only to orient His-57 His-57 acts as a general acid and base Ser-195 forms a covalent bond with peptide to be cleaved Covalent bond formation turns a trigonal C into a tetrahedral C The tetrahedral oxyanion intermediate is stabilized by N-Hs of Gly-193 and Ser-195

R
+

O C N H

R C H

O
C N H

R C H

O C N H

R C H

O
C

H3N

C H

HO CH2 Enz serine residue

R
+

O C

O O CH2 Enz

O C N H

R C H

O
C

N C C H H acyl-enzyme intermediate

H3N

C H

+ H2N

C H

During catalysis, there is nucleophilic attack of the hydroxyl O of a serine residue of the protease on the carbonyl C of the peptide bond that is to be cleaved.

An acyl-enzyme intermediate is transiently formed.

In this diagram a small peptide is shown being cleaved, while the usual substrate would be a larger polypeptide.

R
+

O C N H

R C H

O
C N H

R C H

O C N H

R C H

O
C

H3 N

C H

HO CH2 Enz serine residue

R
+

O C

O O CH2 Enz

O C N H

R C H

O
C

N C C H H acyl-enzyme intermediate R O C N H R C H

H3 N

C H

+ H2N

C H

H2O O
C
+

H3N

C H

OH + HO

CH2

Enz

Hydrolysis of the ester linkage yields the second peptide product.

PDB 3BTK
The active site in each serine protease includes a serine residue, a histidine residue, & an aspartate residue.

Asp102 His57 Ser195

Catalytic residues in trypsin

During attack of the serine hydroxyl oxygen, a proton is transferred from the serine hydroxyl to the imidazole ring of the histidine, as the adjacent aspartate carboxyl is H-bonded to the histidine.

Chymotrypsin
Chymotrypsin is one of the serine proteases. Chymotrypsin is selective for peptide bonds with aromatic or large hydrophobic side chains, such as Tyr, Trp, Phe and Met, which are on the carboxyl side of this bond. It can also catalyze the hydrolysis of easter bond. The main catalytic driving force for Chymotrypsin is the set of three amino acid known as catalytic triad. This catalytic pocket is found in the whole serine protease family.

Properties of an Active Site

A shape that fits a specific substrate or substrates only Side chains that attract the enzyme particular substrate Side chains specifically positioned to speed the reaction

The Catalytic Triad

Chymotrypsin Protein Hydrolysis

Stage #1

Chymotrypsin Protein Hydrolysis

Stage #2

Chymotrypsin Protein Hydrolysis

Stage #4

Chymotrypsin Protein Hydrolysis

Stage #5

Chymotrypsin Protein Hydrolysis

Stage #3

Chymotrypsin Protein Hydrolysis

Stage #6

Transition State Stabilization

Chymotrypsin Kinetics

The initial "burst" in chymotrypsin-catalysed hydrolysis of the p-nitrophenyl acetate