Enzymes are believed to utilize binding energy from the interaction of nonreacting groups of specific substrates with the active site to bring about rate increases. As with substrate binding, covalent bond rearrangement steps in an enzyme reaction sequence also involve energy changes and the question arises as to the role the covalent transformations play in the process of catalysis. It is the purpose of this proposal to concentrate on those steps in the catalytic sequence that involve covalent bond changes and in particular, to characterize covalently modified enzyme intermediates (EA) in terms of covalent and noncovalent Gibbs energy contributions. The research is developed along two approaches, one of which uses a highly strained cyclic ester substrate for Alpha-chymotrypsin and thereby introduces additional energy considerations in the transformations involving this EA species. Structure-activity studies involving the decomposition of this EA species are proposed along with a thermodynamic analysis (DeltaG, DeltaH, DeltaS) of the formation and decomposition of this intermediate. The other approach focuses on obtaining the noncovalent energy contents of enzymes (E) and their covalent intermediates (EA). The noncovalent energy inherent in the protein structures can be determined from the Gibbs energy change (DeltaGH20N leads to RC) for disruption of all intramolecular interactions, i.e. by denaturation of E or EA from their native structures to a random coil. From knowledge of (DeltaGH20N leads to RC) for E and EA along with the Gibbs energy change for E+S to EA, a scheme is provided to calculate the Gibbs energy change for covalent rearrangement and to separate this DeltaG from the noncovalent contributions in the catalytic sequence. Enzymes to be studied include Alpha-chymotrypsin, Staph. aureus penicillinase, and acid phosphatase.
