ROLE OF SERINE-214 AND TYROSINE-171, COMPONENTS OF THE S2 SUBSITE OF ALPHA-LYTIC PROTEASE, IN CATALYSIS

The function of a hydrogen bond network, comprised of the hydroxyl groups of Tyr 171 and Ser 214, in the hydrophobic S2 subsite of alpha-lytic protease, was investigated by mutagenesis and the kinetics of a substrate analog series. To study the catalytic role of the Tyr 171 and Ser 214 hydroxyl groups, Tyr 171 was converted to phenylalanine (Y171F) and Ser 214 to alanine (S214A). The double mutant (Y171F: S214A) also was generated. The single S214A and double Y171F:S214A mutations cause differential effects on catalysis and proenzyme processing. For S214A, k(cat)/K(m) is (4.9 X 10(3))-fold lower than that of wild type and proenzyme processing is blocked. For the double mutant (Y171F:S214A), k(cat)/K(m) is 82-fold lower than that of wild type and proenzyme processing occurs. In Y171F, k(cat)/K(m) is 34-fold lower than that of wild type, and the proenzyme is processed. The data indicate that Ser 214, although conserved among serine proteases and hydrogen bonded to the catalytic triad [Brayer, G. D., Delbaere, L. T. J., & James, M. N. G. (1979) J. Mol. Biol. 131, 743], is not essential for catalytic function in alpha-lytic protease. A substrate series (in which peptide length is varied) established that the mutations (Y171F and Y171F: S214A) do not alter enzyme-substrate interactions in subsites other than S2. The pH dependence of k(cat)/K(m) for Y171F and Y171F:S214A has changed less than 0.5 unit from that of wild type; this suggests the catalytic triad is unperturbed. In wild type, hydrophobic interactions at S2 increase k(cat)/K(m) by up to (1.2 X 10(3))-fold with no effect on K(m). The consequence of the S2 hydroxyl group mutations is to destabilize hydrophobic binding in the S2 subsite. In Y171F and Y171F:S214A, the k(cat)/K(m) values for good dipeptide substrates (containing hydrophobic P2 side chains) decrease 25-45-fold compared to wild type, while k.tl K(m) for poor substrates is unaffected. We conclude the Tyr 171 and Ser 214 hydroxyls do not interact directly with the substrate, but facilitate catalysis by maintaining S2 in an optimal and exact structure.