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Enzymatic Hydrolysis and Group Transfer Reactions 93
(a)
(b)
Figure 5.12 Comparison of the active sites of: (a) thermolysin (PDB Wle 5TMN, active site Glu-143);
(b) carboxypeptidase A (PDB Wle 2CTC, active site Glu-270). In each structure the active site
glutamic acid residue is (on left hand side of each figure) highlighted in red. The Zn 2þ cofactor and
its ligands are illustrated. A bound substrate analogue is drawn in black.
ate using Glu-143 to transfer a proton to the departing nitrogen gives the
hydrolysis products.
In the active site of carboxypeptidase A, the active site Glu-270 is positioned
somewhat closer to the substrate, 2.5 Å away from the amide carbonyl (see
Figure 5.12b), close enough to act either as a nucleophile or as a base. Evidence
for the existence of an anhydride intermediate, formed by nucleophilic attack of
Glu-270 on the amide substrate, has come from the treatment of carboxypeptidase
A with NaB(CN) 3 H 3 in the presence of substrate leading to the incorporation
of 3 H label into the protein and the isolation of 3 H-hydroxynorvaline in
the modiWed enzyme (see Figure 5.13). Further support for an anhydride
intermediate in the carboxypeptidase A reaction has also emerged from resonance
Raman spectroscopic studies of the enzyme-catalysed reaction, showing
bands at 1700 --1800 cm 1 characteristic of anhydrides. Nucleophilic and basecatalysed
mechanisms for carboxypeptidase A and thermolysin, respectively,
are shown in Figure 5.14.
Thermolysin is strongly inhibited (K i 28 nm) by a natural product phosphoramidon,
a monosaccharide derivative containing a phosphonamidate
functional group (Figure 5.15). The phosphonamidate group binds to the
peptide
Glu 270
O
O
O
NaB(CN) 3 H 3
Enz
3 H 3 H
OH
6M HCl
CO 2 H
+
H 3 N
3 H 3 H
3 H-norvaline
OH
anhydride intermediate
Figure 5.13 Sodium cyanoborohydride trapping of putative anhydride intermediate.