Effect of Acylation on Peptide Synthesis by Immobilized Thermolysin

Abstract

KEY WORDSImmobilized Enzyme / Thermolysin / Acylation / Chemical Modification / Peptide Synthesis / A natural enzyme often shows strict specificity so that it is hard to use for a reaction of less specific substrates. Modulation of the enzyme function through structure, by either genetic or chemical procedure may eliminate this difficulty. A number of studies on chemical modification of enzyme has been performed to elucidate the detailed reaction mechanism and the results are very interesting for practical use of enzyme reactions. One example is the peptide formation catalyzed by a proteolytic enzyme. Here we take thermolysin, a thermostable neutral protease from B. thermoproteoliticus, 1 as the target. This enzyme can catalyze a condensation of N-acylamino acid and amino acid amide (eq 1: AC, acylgroup; AA and AA', amino acid residue). 2 AC-AA+AA'-NH 2 ~AC-AA-AA'-NH2 (1) We have investigated several kinetic aspects of free and immobilized thermolysin. 3 • 4 On the mechanism of this enzyme Vallee's school reported extensive studies 5 -7 and showed that this enzyme exhibits different catalytic behavior when the active site zinc ion is replaced by cobalt ion 7 or when Tyr-110 was acylated with N-acetylamino acid. 6 The latter result * To whom all correspondence should be addressed.was further certified by another independent group. 8 Though a peptide condensation reaction is the reverse reaction of hydrolysis, there are many factors controlling the rate and yield and therefore the effect of chemical modification on the condensation reaction cannot be estimated straightforward from the results of hydrolytic reactions.Recently, studies on the effect of metal substitution of thermolysin on the peptide synthesis were reported for the free (soluble) and immobilized states of an enzyme. 9 • 10 Here we examine the effect of acylation on a few simple condensation reactions catalyzed by thermolysin immobilized on glass matrices. The immobilized state is chosen for ease of various chemical treatments and feasibility of applications. Since immobilization of an enzyme is already a "chemical" modification, this example is a sort of double modification.