Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

84 Chapter 5
non-speciWc protease called bromelain present in fresh pineapple, which is why
fresh pineapple will attack your gums if you do not brush your teeth!
There are four main classes of peptidase enzyme, classiWed according to the
groups found at their active site which carry out catalysis. They are as follows:
the serine proteases; the cysteine proteases; the metalloproteases and the aspartyl
(or acid) proteases. Table 5.2 shows the more common commercially available
proteases. The table shows to which mechanistic group they belong,
whether they are exo- orendo-proteases, and what is their preferred site of
cleavage. We shall now consider each class of protease in turn.
The serine proteases
These enzymes are characterised by an active site serine residue which participates
covalently in catalysis. The active site serine is assisted in catalysis by a
histidine and an aspartate residue, the three residues acting in concert as a
‘catalytic triad’. The best characterised of the serine proteases is a-chymotrypsin,
a 241-amino acid endoprotease which shows speciWcity for cleavage after
aromatic amino acids (phenylalanine, tyrosine or tryptophan). This selectivity
arises from a favourable hydrophobic interaction between the aromatic side
chain of the substrate and a hydrophobic binding pocket situated close to the
catalytic site, as illustrated in Figure 5.3.
The catalytic mechanism of chymotrypsin is shown in Figure 5.4. It is a
classic example of covalent catalysis, in which the active site Ser-195 attacks the
amide carbonyl to form a tetrahedral oxyanion intermediate. Attack of Ser-195
is made possible by base catalysis from His-57, generating an imidazolium
cation which is stabilised by the carboxylate of Asp-102. Selective stabilisation
of the high energy oxyanion intermediate takes place via formation of two
peptide-NH
R
O
H
N
R'
O
NH-peptide
- specific for L-amino acids (or D- in a few cases)
- usually specific for amino acid preceding cleavage site.
α-chymotrypsin
- cuts after Phe, Tyr, Trp
(aromatic side chains)
trypsin
- cuts after Arg, Lys
(basic side chains)
O
H
N
O
N
H
O
H
N
O
N
H
H 3 N
+
O O −
Figure 5.3 SpeciWcity of endopeptidases.