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In the zinc metallopeptidases produced by the genus Bacillus, an active site histidine has been proposed to
either stabilize the transition state in catalysis by donating a
hydrogen bond to the hydrated peptide (Matthews, B. W.(1988) Acc. Chem. Res. 21, 333-340) or to polarize a water
molecule, which subsequently attacks the peptidyl bond (Mock, W. L.,
and Aksamawati, M.(1994) Biochem. J. 302, 57-68).
Site-directed mutagenesis techniques have been used to change this
residue in the zinc endopeptidase from Bacillus
stearothermophillus to either phenylalanine or alanine. At pH 7.0,
the k
Volume 270,
Number 28,
Issue of July 14, pp. 16803-16808, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A SITE-DIRECTED MUTAGENESIS STUDY
/K
values of
the substrate leucine enkephalin for the phenylalanine and alanine
mutants were reduced by factors of 430- and 500-fold, respectively, as
compared with the wild-type enzyme, mostly due to changes in k
. In addition, the enzymatic activities of the
mutant enzymes showed little pH dependence in the alkaline range,
unlike the wild-type enzyme. The mutations did not greatly alter the
binding affinities of inhibitors containing sulfydryl groups to chelate
the active site zinc, while those of inhibitors containing hydroxamate
or carboxylate zinc-chelating groups were increased between 80- and
250-fold. The largest change in the binding affinity of an inhibitor
(>5 orders of magnitude) was found with the proposed transition
state mimic, phosphoramidon. The results are generally in agreement
with x-ray crystallography studies and favor the involvement of the
active site histidine in transition state binding.
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