Effect of Boric Acid on the Catalytic Activity of Streptomyces griseus Protease 3

Abstract

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 a t 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 pK, 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 chymotry5sin as concerns the structure and function of the catalytic site.The hydrolytic activity of the serine enzyme Streptomyces griseus protease 3 exhibits in the absence of boric acid a pH-dependence closely similar to that of pancreatic serine proteases [I], which suggests that the anomalous pH-dependence observed for the bacterial enzyme in borate-containing buffer systems [2] can be attributed to effects of boric acid on the catalytic activity of the protein. Boric acid and boronic acid derivatives have been reported to act as transition-state analogues for serine hydrolases such as chymotrypsin [3-71 and subtilisin [7,8], inhibiting enzyme activity by binding to the active site serine under formation of a tetrahedral adduct, and a similar mechanism of enzyme inhibition might be operative in the interaction between boric acid and Streptomyces griseus protease 3. I n order to test this idea, and hence to obtain further information about the detailed mechanistical similarities or dissimilarities between the bacterial enzyme and pancreatic serine proteases, the inhibitory effect of boric acid on the steady-state and presteady-state rate-behaviour of Xtreptomyces griseus protease 3 has been investigated and analyzed.EXPERIMENTAL PROCEDURE Materials and methods used in the present investigation were the same as those described in the precceding pape...

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