Some Properties of Leucine Aminopeptidase from Aspergillus japonica as a Metalloenzyme

Abstract

Some properties of purified leucine aminopeptidase [E.C. 3.4.1.1] from Aspergillus japonica as a metalloenzyme were investigated. Leucine aminopeptidase was inhibited by various chelating agents. The addition of Zn²⁺ to the ethylenediaminetetraacetic acid (EDTA)-treated leucine aminopeptidase restored the activity to nearly the original level, but Co²⁺ was partially effective. Kinetic studies using L-leucyl-β-naphthylamide as substrate were carried out with native leucine aminopeptidase, Zn²⁺ or Co²⁺-reactivated leucine aminopeptidase after dialysis against EDTA. Michaelis constant (K_m) and activation energy (E_act) of native leucine aminopeptidase were in good agreement with those of Zn²⁺ reactivated leucine aminopeptidase (native leucine aminopeptidase K_m : 2.5×10^-4 M, E_act : 9.2×10³ cal/mol, Zn²⁺-reactivated leucine aminopeptidase K_m : 2.5×10^-4 M, E_act : 9.8×10³ cal/mol). In the presence of L-leucyl-β-naphthylamide, the inhibition of leucine aminopeptidase by various chelating agents and a Cd²⁺ decreased to an extent of 23-46%. Metal analysis indicated that the purified leucine aminopeptidase containing 1 g-atom of zinc per mol of leucine aminopeptidase, and both the activity and the zinc content were lowered when native leucine aminopeptidase was treated with EDTA or o-phenanthroline.