Made for 4th year class

1. Serine Proteases

2. serine (Ser, S)
H
H3N+ C COO
CH2
OH
Serine proteases include digestive
enzymes trypsin, chymotrypsin, & elastase.
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.

3. 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

4. R O R O R O R O
+
H3N C C N C C N C C N C C O + HO CH2 Enz
H H H H H H H serine residue
R O R O R O R O
+
H3N C C N C C O CH2 Enz + H2N C C N C C O
H H H H H H
acyl-enzyme intermediate
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.

5. R O R O R O R O
+
H3 N C C N C C N C C N C C O + HO CH2 Enz
H H H H H H H serine residue
R O R O R O R O
+
H3 N C C N C C O CH2 Enz + H2N C C N C C O
H H H H H H
acyl-enzyme intermediate
H2O
R O R O
+
H3 N C C N C C OH + HO CH2 Enz
H H H
Hydrolysis of the ester linkage yields the second peptide
product.

6. PDB 3BTK
The active site in each serine
Asp102
protease includes a serine residue, a His57
histidine residue, & an aspartate
residue.
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.

8. 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.

9. 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

10. The Catalytic Triad

12. Chymotrypsin Protein Hydrolysis
Stage #1

13. Chymotrypsin Protein Hydrolysis
Stage #2

14. Chymotrypsin Protein Hydrolysis
Stage #4

15. Chymotrypsin Protein Hydrolysis
Stage #5

16. Chymotrypsin Protein Hydrolysis
Stage #3

17. Chymotrypsin Protein Hydrolysis
Stage #6

18. Transition State Stabilization

20. Chymotrypsin Kinetics
The initial "burst" in
chymotrypsin-catalysed
hydrolysis of the p-nitrophenyl
acetate