THE ROLE OF HISTIDINE-231 IN THERMOLYSIN-LIKE ENZYMES - A SITE-DIRECTED MUTAGENESIS STUDY

In the zinc metallopeptidases produced by the genus Bacillus, an activ e 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 z inc endopeptidase from Bacillus stearothermophillus to either phenylal anine or alanine. At pH 7.0, the k(cat)/K-m values of the substrate le ucine enkephalin for the phenylalanine and alanine mutants were reduce d by factors of 430- and 500-fold, respectively, as compared with the wild-type enzyme, mostly due to changes in k(cat). In addition, the en zymatic activities of the mutant enzymes showed little pH dependence i n the alkaline range, unlike the wild-type enzyme. The mutations did n ot greatly alter the binding affinities of inhibitors containing sulfy dryl groups to chelate the active site zinc, while those of inhibitors containing hydroxamate or carboxylate zinc-chelating groups were incr eased between 80- and 250-fold. The largest change in the binding affi nity of an inhibitor (>5 orders of magnitude) was found with the propo sed transition state mimic, phosphoramidon. The results are generally in agreement with x-ray crystallography studies and favor the involvem ent of the active site histidine in transition state binding.