Effect of pH on the catalytic activity of Streptomyces griseus protease 3.

Abstract

The kinetics of the Streptomyces griseus protease-3-catalyzed hydrolysis of the non-specific ester substrate p-nitrophenylacetate and the specific peptide substrate glutaryl-l-phenylalanine-p-nitroanilide are consistent with a three-step mechanism of Michaelis complex formation followed by enzyme acylation and deacylation. Both substrates pre-equilibrate rapidly with the free enzyme, the acylation (deacylation) step being rate-limiting in the enzymatic hydrolysis of glutaryl-l-phenylalanine-p-nitroanilide (p-nitrophenylacetate). Examination of the effect of pH on steady-state and transient-state kinetic parameters for the enzymatic hydrolysis of the two substrates provides evidence that the stability of the Michaelis complex is essentially unaffected by pH over the range investigated, whereas both acylation and deacylation of the enzyme is dependent upon an ionizing group in the protein with apparent pKa of 6.6. It is concluded that closely similar mechanisms of activation of the reactive seryl residue may be operative in Streptomyces griseus protease 3 and pancreatic serine proteases such as chymotrypsin and elastase.