Kinetic characterization of alkaline mesentericopeptidase. Comparison with serine proteinases from different origins.

Abstract

Comparative studies of the hydrolysis of succinyl-Ala2-Phe-methylcoumarylamide with mesentericopeptidase, a mesophilic extracellular serine proteinase from Bacillus mesentericus, and proteinases produced by organisms representing different levels of evolutionary development, were performed. Drastic differences in the proteolytic coefficient kcat/Km were found. As regards their catalytic efficiency, the proteinases studied can be placed in the following order: mesentericopeptidase < subtilisin Novo << subtilisin DY < proteinase K < subtilisin Carlsberg < thermitase < alpha-chymotrypsin. The size of the substrate-binding site of mesentericopeptidase for synthetic peptides was studied by using chloromethyl ketones with the general formula benzyloxycarbonyl-Alan-Phe-CH2Cl (n = 1, 2, 3). The presence of at least five binding subsites (S1 ... S5) on the S-side of the hydrolysed bond was suggested. Studies of the primary specificity of mesentericopeptidase with a series of dipeptide chloromethyl ketones having the general formula benzyloxycarbonyl-Ala-Aa-CH2Cl (Aa = Ala, Val, Leu, Phe) revealed the following order of reactivity toward these inhibitors: Aa = Leu >> Ala > Phe > Val. Kinetically, mesentericopeptidase is similar to subtilisin BPN'/Novo.