An intracellular aminopeptidase from Streptomyces rimosus that prefers basic amino acids

Abstract

An aminopeptidase from the mycelia of Streptomyces rimosus was isolated in an electrophoretically homogeneous form. It was shown to be a monomeric, acidic protein (pI = 4.4, mol. wt. approx. 83,000), with optimal activity at pH 7.1–7.8 and at 35–41° C. The enzyme was fully inhibited by 0.1 mM EDTA or 1 mM o-phenanthroline; the activity was restored upon addition of 0.05 mM Co²⁺, Zn²⁺, or Ni²⁺. Amastatin, bestatin, and puromycin also inhibited the enzyme. The aminopeptidase hydrolyzed amino-acid-2-naphthylamides and various di- to heptapeptides. The highest catalytic coefficients (23 and 19 μM^–1 s^–1) were obtained with Arg- and Lys-2-naphthylamide, followed by Leu-, Phe- and Met-derivatives with one order of magnitude lower catalytic coefficients. Basic or bulky hydrophobic amino acids at the P₁ and/or P₁′ position of peptide substrates were preferred. Acidic amino acids and proline were not accepted. The affinity of the enzyme increased with the length of peptide. According to these properties, S. rimosus intracellular aminopeptidase is distinct from the extracellular leucine aminopeptidase of the same organism and can be classified as an Arg(Lys)-preferring metalloaminopeptidase.