Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

92 Chapter 5
now predominate Perhaps, as mentioned above, because they are more stable
in an oxygen atmosphere.
The metalloproteases
The metalloproteases are characterised by a requirement for an active site
metal ion, usually Zn 2þ , which is involved in the catalytic cycle. These enzymes
can be readily distinguished from the other classes by treatment with metal
chelating agents such as ethylene diamine tetra-acetic acid (EDTA) or
1,10-phenanthroline (see Figure 5.11), leading to removal of the metal ion
cofactor and inactivation.
The best characterised members of this family are carboxypeptidase
A, a 307-amino acid exopeptidase from bovine pancreas which cleaves the
C-terminal residue of a peptide chain (not arginine, lysine or proline); and
thermolysin, a 35-kDa endopeptidase from Bacillus thermoproteolyticus which
cleaves before hydrophobic amino acids such as leucine, isoleucine, valine or
phenylalanine. Both enzymes contain a single Zn 2þ ion at their active sites,
which in the resting state of the enzyme is co-ordinated by three protein ligands
and one solvent water molecule. There is evidence to suggest that in both
enzymes when the substrate is bound the water molecule is displaced by the
carbonyl oxygen of the amide bond to be hydrolysed, which is thus activated
towards nucleophilic attack by Zn 2þ Lewis acid catalysis.
The mechanism of amide bond hydrolysis has been well studied in both
enzymes, with slightly diVerent results emerging. Both enzymes contain an
active site glutamate, which in theory could either act as a nucleophile to attack
the amide carbonyl, or act as a base to deprotonate an attacking water molecule.
In thermolysin the active site glutamate (Glu-143) is positioned 3.9 Å
away from the amide carbonyl (see Figure 5.12a), too far away to act as
a nucleophile, but far enough to accommodate an intervening water molecule,
which is thought to be activated by co-ordination to the Zn 2þ cofactor. There is
evidence from X-ray crystallography to suggest that Glu-143 acts as a base to
deprotonate an attacking water molecule, forming an oxyanion intermediate
which is stabilised by speciWc hydrogen bonds. Breakdown of this intermedi-
O
O
N N
−
O
O
−
M
n+
−
O
−
O
O
O
N
N
M
n+
Ethylene diamine tetra-acetic acid (EDTA)
1,10-Phenanthroline
Figure 5.11 Metal chelating agents.