Biochemical characterisation of an aminopeptidase with highest preference for lysine from Japanese flounder skeletal muscle

Abstract

An aminopeptidase was purified from Japanese flounder skeletal muscle to homogeneity by ammonium sulphate fractionation and three chromatographies. The enzyme was approximately 100kDa with isoelectric point of 5.7 as estimated by two-dimensional polyacrylamide gel electrophoresis. Its optimum temperature and pH were 45°C and 7.5, respectively. According to peptide mass fingerprinting study, the enzyme revealed high identity to a puromycin-sensitive aminopeptidase. It had a broad specificity toward aminopeptidase substrates and preferred to hydrolyse Lys-MCA with kcat/Km of 8.1×106M−1s−1, and the activation energy (Ea) of 72.5kJM−1. Metal-chelating agents effectively inhibited the enzyme activity, and Zn2+, Mn2+ and Co2+ significantly restored the apoenzymatic activity dialysed by EDTA, whilst inhibitors to other proteinases did not show much effect. Furthermore, bestatin strongly inhibited its activity. These results indicate that the purified enzyme is a metalloaminopeptidase which would possibly contribute to free amino acids increase in fish muscle.