Subsite Mapping of an Acidic Amino Acid-Specific Endopeptidase from Streptomyces griseus, G1uSGP, and Protease V8

Abstract

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, G1uSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitro-anilide substrates which have a Glu residue at the P₁ position. G1uSGP and protease V8 favored Pro and Leu residues at S₂, respectively, while the S₃ subsite of G1uSGP preferred Phe over either Ala or Leu. The S₃ subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for G1uSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a K_m value of 0.41mM (0.1 M Tris-HC1, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a K_m value of 0.25mM (0.1M phosphate, pH 7.8), respectively. The k_cat/K_m values for these substrates obtained with G1uSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.