EFFECT OF PRESSURE ON SUBTILISIN CATALYSIS - HYDROLYSIS AND PEPTIDE-SYNTHESIS

The effects of high pressure on the subtilisin (Carlsberg) catalyzed h ydrolysis and aminolysis reactions were investigated. The pH dependenc e of the apparent second-order rate constant (k(cat)(K-m(app)) for N-g lutaryl-Ala-Ala-Pro-Leu-pNA showed a slightly lowered profile due to i ncreasing pressure. The pressure dependence (up to 150 MPa) of k(cat)/ K-m(app) for this substrate or N-benzyloxycarbonyl-Gly-Gly-Leu-pNA at a constant pH (8.5) showed slightly positive activation volumes: + 2-3 ml mol(-1). The substrate concentration dependence of the hydrolytic rate for N-succinyl-Ala-Ala-Pro-Phe-pNA showed increased slope and Y-i ntercept on an Eadie plot at 150 MPa; the reaction volume for the 1/K- m was positive and the activation volume for k(cat) was estimated to b e negative. Pressure affected the two parameters in the opposite direc tions,and as a result, the effect on K-cat/K-m(app) became rather smal l. Pressure promoted the stubilisin-catalyzed transacylation reaction between Fua-Phe-OMe and GlyNH(2), but the fraction of the peptide form ation (f(a)) decreased slightly under high pressure. Pressure seemed t o accelerate the hydrolytic process much more, especially the nucleoph ile-assisted hydrolysis of the acyl-enzyme, than the acyl-transfer to the nucleophile.