Covalent bond changes as a driving force in enzyme catalysis.

Abstract

A procedure is developed for assessing covalent and noncovalent aspects of the acylation of alpha-chymotrypsin (alpha-Ct) by the substrate trifluoroethyl furoate (S) to form furoyl-chymotrypsin (F-Ct) and trifluoroethanol (P1). The free energy change (-4.31 kcal/mol) for the acylation at pH 7, alpha-Ct+S <--> F-Ct+P1, contains contributions from covalent bond changes as well as noncovalent changes. The noncovalent changes are considered to be manifested in the structures of alpha-Ct and F-Ct, and a noncovalent free energy difference between alpha-Ct and F-Ct has been evaluated from the difference in unfolding free energy changes for these proteins. The unfolding free energy changes demonstrate that F-Ct is 1.6 kcal/mol less stable than alpha-Ct at pH7. Thus, despite an overall favorable free energy change for acyl-enzyme formation (-4.31 kcal/mol), covalent linkage of the furoyl moiety to the active site is thermodynamically destabilizing to the enzyme. The consequence of this unfavorable effect of the furoyl moiety on the noncovalent free energy is that "binding" of the covalently linked furoyl moiety cannot be the driving force for acylation. The source of free energy driving acylation is the covalent bond-breaking and bond-making involved in transesterification of the furoyl group to the enzyme. Since the (noncovalent) Michaelis complex between alpha-Ct and substrate is significantly more stable (thermodynamically) than F-Ct, a substantial amount of noncovalent free energy must be given up on forming F-Ct. The destabilization residing in F-Ct is consistent with the possibility that energy transduction occurs when the Michaelis complex is converted to F-Ct and that destabilization is relieved on reaching the next activated complex.