Specificity of Streptomyces griseus aminopeptidase and modulation of activity by divalent metal ion binding and substitution

Abstract

Streptomyces griseus aminopeptidase is a calcium‐activated zinc metalloenzyme characterized by a high enzymic reactivity, high thermal stability and low molecular mass [Spungin, A. and Blumberg, S. (1989) Eur. J. Biochem. 183, 471–477]. A study of the specificity of S. griseus aminopeptidase using amino acid 4‐nitroanilide substrates shows that the leucine derivative is the best substrate. Derivatives of other hydrophobic amino acids, methionine and phenylalanine, are also excellent substrates for the enzyme. The 4‐nitroanilides of alanine, valine, proline and lysine are good substrates whereas those of the small size glycine and the acidic amino acids are very poor. No hydrolysis of a terminal Xaa residue can be detected with Xaa‐proline N‐terminal sequences. Calcium ions bind to the enzyme and modulate its activity in a substrate‐dependent manner. The catalytically essential zinc of S. griseus aminopeptidase is removed by dialysis against 1,10‐phenanthroline and replaced by manganese or cobalt ions, resulting in enzyme derivatives of altered specificities. Thus, whereas the zinc enzyme hydrolyzes leucine 4‐nitroanilide at a 10‐fold faster rate than the manganese or cobalt enzymes, the cobalt enzyme hydrolyzes alanine 4‐nitroanilide at a more than 20‐fold faster rate than the zinc or manganese enzymes. Copyright © 1993, Wiley Blackwell. All rights reserved