Specificity and mode of action of acid carboxypeptidase from Aspergillus saitoi

Abstract

1. 1. Acid carboxypeptidase isolated from culture filtrate of Aspergillus saitoi has been investigated for its use in carboxyterminal sequence determination of native insulin, angiotensin I and glucagon at pH 2.5 or 2.2. The examination indicated that after 5 h incubation asparagine, alanine and lysine were liberated from native insulin. The acid carboxypeptidase catalyzed the release of leucine and histidine from the decapeptide angiotensin I. When (Pro-ProGly) 5 and polylysine were incubated separately with the acid carboxypeptidase, no hydrolysis was found. No release of free amino acids by autodigestion of the enzyme was detected after a prolonged period of incubation. 2. 2. The pH dependene of log ( V/ K m ) and log V for the acid carboxypeptidase-catalyzed hydrolysis of a small synthetic peptide, Z-Tyr-Leu, has been determined. Bell-shaped pH-rate profiles were obtained. The main conclusions derived from analysis of the data were that two catalytically-active groups on the enzyme with p K e1 ≅ 2.3 and p K e2 ≅ 4.9 were important in the enzymatic action at the acid end of the pH range. Competitive inhibition by small substrates was found with hydrocinnamic acid, indole-3-propionic acid and 4-phenylbutyric acid. The K i value for hydrocinnamic acid inhibition was 4·10 −4 M.