Effect of Boric Acid on the Catalytic Activity of <em>Streptomyces griseus</em> Protease 3.

1. Boric acid acts as a competitive inhibitor of the Streptomyces griseus protease-3-catalyzed hydrolysis of p-nitrophenylacetate, indicating that there is no significant interaction between inhibitor and the acyl-enzyme formed during the reaction. 2. Boric acid affects the rate of the transient burst of p-nitrophenol formation from p-nitrophenylacetate in a way suggesting that the inhibitor binds equally firmly to the free enzyme and the Michaelis complex formed between enzyme and substrate. The same conclusion can be drawn from the observation that boric acid acts as a non-competitive inhibitor of the enzymatic hydrolysis of glutaryl-L-phenylalanine-p-nitroanilide. 3. Apparent equilibrium constants for the binding of boric acid to the free enzyme and the Michaelis complex have been determined kinetically at different pH between 5 and 10. The data obtained provide evidence that the stability of the enzyme, inhibitor complex is dependent upon an ionizing group in the protein with apparent pKa of 6.6, and further show that there is no interaction between enzyme and the ionized form of the inhibitor. 4. The above results suggest that boric acid has no effect on the process of Michaelis-complex formation, but inhibits enzyme activity by binding covalently to the active-site seryl residue under formation of a tetrahedral adduct, which might represent a transition-state analogue for the enzymatic conversion of bound substrate into bound product. It is concluded that the nucleophilic reactivity of the active-site seryl residue is unaffected by the binding of p-nitrophenylacetate to the protein, and that Streptomyces griseus protease 3 may be considered as homologous with chymotrypsin as concerns the structure and function of the catalytic site. [ABSTRACT FROM AUTHOR]