Binding of amino acid side-chains to S-1 cavities of serine proteinases

The P-1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S-1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P-1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P-1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (+/-2)degrees C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S-1 cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin A alpha (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with K-a values for five non-coded variants at P-1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest K-a for each of the six enzymes range from 10(6) to 10(8). The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and ne), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu(-) and Asp(-) are strongly unfavorable. The Pearson product moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P-1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (-0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity. (C) 1997 Academic Press Limited.