Equilibriums of Bowman-Birk inhibitor association with trypsin and α-chymotrypsin

Abstract

Association constants, enthalpies, and stoichiometries of Bowman-Birk soybean inhibitor for trypsin and alpha-chymotrypsin were measured in the pH range 4-8 at 25 degrees, 0.01 M Ca2+. The results are quoted in terms of moles of protease active sites, from active site titration. Enthalpies were obtained from calorimetry. The inhibitor was modified by carboxyl group modification, and by tryptic and chymotryptic attack. Association thermodynamics and stoichiometries of the modified inhibitors with both proteases were also determined. There is one independent site for each protease on the inhibitor protein. Modification decreases association to some extent, but does not appear to change stoichiometry or protease binding site independency. In the pH 4 region the association enthalpies are endothermic, of the order 6 kcal/mol for both trypsin and chymotrypsin. With increasing pH, the enthalpies decrease and become exothermic at pH 8 for chymotrypsin. Positive entropies, 50 cal mol-1 deg-1, occur at pH 4-5. They decrease as pH increases, but are always positive in sign. The observed to accompany the overall reaction, such as H+ transfer steps. The enthalpies and entropies probably compensate over the pH range 4-8, with a characteristic temperature of 390 plus or minus 30 degrees K. Estimates were made of the macromolecular Coulomb charge products in inhibitor-protease interaction. These range from about +5 to -60, over pH range 4-8, depending on the protease. Although intermolecular Coulombic forces cannot be easily delineated at the specific side chain level, they may operate at the macromolecule level.

Template-Directed Protein Folding into a Metastable State of Increased Activity

“…2a) and the correct ratio of the two subdomains is not affected after prolonged incubation in the refolding buffer (Fig. The K, values of the subdomains are in close accord with the properties of the natural soybean protein (Turner et al, 1975). The K, values of the subdomains are in close accord with the properties of the natural soybean protein (Turner et al, 1975).…”

Dissecting the energetics of a protein-protein interaction: the binding of ovomucoid third domain to elastase 1 1Edited by P. E. Wright

“…It seems a general feature of these inhibitors that binding results in a pK a shift for His57. While the in¯uence of this proton linkage on the energetics has been noted (Turner et al, 1975;Barnhill & Trowbridge, 1975;Yung & Trowbridge, 1980), the intrinsic energetics have not been determined in these studies. Additionally, the values reported by Ascenzi and co-workers have been obtained from van't Hoff analyses, but the contribution from the large heat capacity change expected for these interactions has not been taken into account (e.g.…”

“…Data are available for Kazal inhibitors (Menegatti et al, 1987;Ascenzi et al, 1991b;Schweitz et al, 1973;Baugh & Trowbridge, 1972), Kunitz inhibitors (Ascenzi et al, 1990a,b;Baugh & Trowbridge, 1972; and inhibitors belonging to other families (Ascenzi et al, 1991a(Ascenzi et al, , 1995Turner et al, 1975). It seems a general feature of these inhibitors that binding results in a pK a shift for His57.…”

Thermodynamic measurements on the interaction of porcine trypsin with single- and two-chain trypsin inhibitors from corn seeds

“…Considering the results involving the single-chain inhibitor in cacodylate and PIPES buffers and that cacodylic acid and PIPES have rather different values for A.Hionization (0.3 and -3 keal/mol, respectively, under our conditions), we further conclude that essentially no protons were taken up by the buffer when trypsin reacted with this form of the inhibitor. The most recent and thorough calorimetric investigations of the reactions of trypsin inhibitors with trypsin were with the Bowman-Birk inhibitor (Turner et al, 1975) and the Kunitz inhibitor (Barnhill and Trowbridge, 1975) from soybeans. Corn trypsin inhibitor is quite different structurally from both of these proteins.…”

“…In the substantial body of investigation on trypsin inhibitors relatively little attention has been given to thermodynamics, even though a thorough grasp of the energetics will be essential to fully understand the nature of the binding reactions. Calorimetric studies have been reported on the interactions of bovine trypsin with the Kunitz and Bowman-Birk inhibitors isolated from soybeans (Turner et al, 1975;Barnhill and Trowbridge, 1975). Here we report the results from a study of the association reactions of porcine trypsin with the single-and two-chain forms of corn trypsin inhibitor, a protein that is quite different structurally from both of the soybean inhibitors (Swartz et al, 1977).…”