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 primar y specificity cavity of the cognate enzyme upon enzyme-inhibitor compl ex formation. Both natural evolution and protein engineering often cha nge the P-1 residue to greatly alter the specificity and the binding s trength. To systematize such results we have obtained all 20 coded P-1 variants of one such inhibitor, turkey ovomucoid third domain, by rec ombinant 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 characte rized serine proteinases with hydrophobic S-1 cavities. The enzyme nam es 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), S treptomyces 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 ovomucoi d third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest K-a for each of the six enzyme s range from 10(6) to 10(8). The dominant force for binding to these p ockets is the hydrophobic interaction. Excess steric bulk (too large f or 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 correlati ons for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enz ymes at P-1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, c hymotrypsin 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 b inding specificity. (C) 1997 Academic Press Limited.