Thermodynamics and kinetics of single residue replacements in avian ovomucoid third domains: effect on inhibitor interactions with serine proteinases

Abstract

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.

Calculation of the free energy of association for protein complexes

“…Interestingly, using volumes to obtain ASP values (cal/A3) produces the best correlation, suggesting that cavity formation is an important parameter in calculating the free The experimental association energies were obtained from the literature. a, Vincent and Lazdunski (1972); b, Vincent et al (1974); c, Chen and Bode (1983); d, Hass and Ryan (1980); e, Bunting and Myers (1975); f, Read et al (1983); g, Ascenzi et al (1988); h, Empie and Laskowski (1982); i, Bode (1979) and Bolognes et al (1982); j , Pekar and Frank (1972); k , Akasaka et al (1982); I, Sheriff et al (1987); m, Ross et al (1977). Chothia and Janin (1975) first suggested that the free energy of association was related to the amount of surface area that was buried in the interface.…”

Heparin Enhances the Specificity of Antithrombin for Thrombin and Factor Xa Independent of the Reactive Center Loop Sequence

“…A hallmark of the lock-and-key mode of complex stabilization is the destabilizing effect of amino acid substitutions in reactive center loop residues of such inhibitors on complex formation, as reflected by increases in the rate constants for complex dissociation (47). Our finding that reactive center loop mutations in antithrombin minimally affect the rate constants for dissociation of antithrombin-proteinase complexes thus supports the idea that reactive center loop interactions with the proteinase active site do not significantly contribute to stabilizing the complexes.…”

The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3)

“…The P3 to P2' residues run as follows: Cys-16, Thr-17, Met-18, Glu-19, Tyr-20. Thus, in contrast to OMJPQ3, which is a trypsin inhibitor because of a lysine residue at its PI position, the methionine of OMSVP3 determines its preferred inhibitory specificity towards chymotrypsin, elastase and subtilisin [7]. OMTKY3 differs from OMSVP3 only by a leucine residue in the PI position resulting in almost identical binding constants.…”