Effects of chelators and metal ions on purified leucine-specific aminopeptidase from aeromonas caviae T-58

Abstract

An extracellular, aminopeptidase (AMP) of a bacterial soil isolate, Aeromonas caviae T-58, was purified to eleclrophorclically homogeneity by ammonium sulfate precipitation and ion-exchange chromatography (Q-Sepharose fast flow and Mono-Q column) to 48-fold with a yield of 3.0%. The purified native enzyme is a monomer and exhibited a single band with molecular weight of 32 kDa estimated by SDS/polyaciylamide-gel electrophoresis. The enzyme was inactivated by Mn2+, Co 2+, Cu2+, and Cd2+, but not affected by Ca2+ Ba2+, Zn2+, AI3+, NI2+, LI2+ Pb2+ and Mg2+. EDTA completely inhibited enzyme activity indicative of the enzyme to a metalloenzyme type. The addition of I mM Zn2+ restored 100% activity of EDTA-inhibited enzyme while I mM Co2+ restored 10% activity. However, the addition of equimolar concentrations of both metals showed a non co- catalytic effect, as residual activity reduced to 90%. The enzyme therefore possibly belongs to a catalytic family of Zn2+ metalloenzyme and does not require Ca2+ for enzymatic activation. The purified enzyme showed a high affinity for L-Leu- p-nitroanilide and valine but not with proline, glycine or alanine-pNA.