Thermodynamics of binding to native α-chymotrypsin and to forms of α-chymotrypsin in which catalytically essential residues are modified; a study of "productive" and "nonproductive" associations

Abstract

The standard free energy (.DELTA.G.degree.), enthalpy (.DELTA.H.degree.), and entropy (.DELTA.S.degree.) of association for proflavin and D- and L-N-AcTrp [L-N-acetyl tryptophan] were obtained at pH 7.8 for native .alpha.-chymotrypsin (Cht) and for forms of Cht in which essential catalytic residues of the active site are modified. The modified Cht forms studied are dehydroalaninyl-195-.alpha.-Cht and N-methylhistidinyl-57-.alpha.-Cht. Associations to native Cht (pH 7.8) are characterized by negative .DELTA.H.degree. and .DELTA.S.degree. values (i.e., for L-AcTrp .DELTA.H.degree. = -9.1 kcal/mol and .DELTA.S.degree. = -21 cu at T = 25.degree. C). Associations to modified Cht are characterized by an enthalpy near zero and a positive entropy of association, the values of the .DELTA.H.degree. and .DELTA.S.degree. for association to the modified Cht forms being similar to those expected for transfer of small aromatic molecules from water to a nonpolar solvent phase. Differences in .DELTA.H.degree. and .DELTA.S.degree. observed for binding of substrate analogues and inhibitors to modified and native Cht (pH 7.8) are approximately +10 kcal/mol and +30 eu, respectively. Data from D. D. F. Shiao similarly show differences of comparable magnitude between binding of substrate analogues to active .alpha.-Cht (pH 7.8) and the His-57 protonated form of .alpha.-Cht (pH 5.6). The negative .DELTA.H.degree. and .DELTA.S.degree. values of associations for binding to active .alpha.-Cht indicate that a substrate-induced conformational change occurs on substrate association with the primary binding site (S1), which does not occur in Ser-195 and His-57 modified Cht. From these differences a linkage between binding of substrate into S1 and the catalytic residues in the nucleophilic subsite (S1-S1''). The data also show that associations of substrate analogues into potentially productive Michaelis complexes S1 cannot be easily differentiated from associations that are nonproductive (i.e., nonactivated) from their .DELTA.G.degree.obsd [observed .DELTA.G.degree.], but may be differentiated by their respective .DELTA.H.degree.obsd and .DELTA.S.degree.obsd for association. The probable substrate association-activation process, characterized thermodynamically in this work, occurs in the substrate binding step and leads to lowered free energies of activation in catalytic steps succeeding binding; the process does not influence the observed strength of substrate binding.