KINETIC CHARACTERIZATION OF ALKALINE MESENTERICOPEPTIDASE - COMPARISON WITH SERINE PROTEINASES FROM DIFFERENT ORIGINS

Comparative studies of the hydrolysis of succinyl-Ala2-Phe-methylcouma rylamide with mesentericopeptidase, a mesophilic extracellular serine proteinase from Bacillus mesentericus, and proteinases produced by org anisms representing different levels of evolutionary development, were performed. Drastic differences in the proteolytic coefficient k(cat)/ K(m) were found. As regards their catalytic efficiency, the proteinase s studied can be placed in the following order: mesentericopeptidase < subtilisin Novo much greater than subtilisin DY < proteinase K < subt ilisin Carlsberg < thermitase < alpha-chymotrypsin. The size of the su bstrate-binding site of mesenterico-peptidase for synthetic peptides w as studied by using chloromethyl ketones with the general formula benz yloxycarbonyl-Ala(n)-Phe-CH2Cl (n = 1, 2, 3). The presence of at least five binding subsites (S1 ... S5) on the S-side of the hydrolysed bon d was suggested. Studies of the primary specificity of mesentericopept idase with a series of dipeptide chloromethyl ketones having the gener al formula benzyloxycarbonyl-Ala-Aa-CH2Cl (Aa = Ala, Val, Leu, Phe) re vealed the following order of reactivity toward these inhibitors: Aa = Leu much greater than Ala> Phe> Val. Kinetically, mesentericopeptidas e is similar to subtilisin BPN'/Novo. (C) Munksgaard 1993.