Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor interactions with serine proteinases.
Sequence determinations in our laboratory have yielded the primary structures of ovomucoid third domains from 35 avian species. From that list, 12 sequences could be arranged into a contiguous set such that each sequence differs from a second by a single amino acid replacement. For this set of domains and for five additional domains of special interest, we report here the association equilibrium constants for their binding with bovine alpha-chymotrypsin, elastase I, and subtilisin Carlsberg. The results are interpreted with the aid of the three-dimensional structure of highly homologous Japanese quail ovomucoid third domain and of computer-generated models of the complexes of the inhibitor with the respective enzymes. The results show that (i) changes in inhibitor residues other than the primary recognition residue (P1) even sequentially far from the reactive site, may exert large effects on association equilibrium constant values provided these residues make contact with the enzyme, (ii) changes in residues other than P1 often exert large differential effects toward the different enzymes, i.e., the same change can make the inhibitor stronger for one enzyme and weaker for another, (iii) the sign and to some extent the magnitude of the changes can be rationalized from the known structures of the inhibitor and the enzyme, (iv) changes in surface residues which do not contact the enzyme in complex are virtually without effect, and (v) glycosylated and nonglycosylated inhibitors have the same constants. For confirmation of the validity of the equilibrium constant comparisons in a few cases, the rate constants kon and kd were determined and the resultant calculated equilibrium constant values compared to the directly determined numbers. An additional test of validity is provided by experiments where a glycosylated domain of one species is allowed to compete with an unglycosylated domain of another for the same enzyme.
D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds / Q000378:metabolism*; D004527:Egg Proteins / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D010059:Ovomucin / Q000378:metabolism / Q000494:pharmacology*; D011480:Protease Inhibitors; D012697:Serine Endopeptidases; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D004527:Egg Proteins; D011480:Protease Inhibitors; D010059:Ovomucin; D010450:Endopeptidases; D012697:Serine Endopeptidases