Protein-ligand interactions as a driving force for a high-enthalpy two-state transition in glutamate dehydrogenase: the opposing roles of phosphate and acetate ions.

It is known that the binding of the reduced coenzyme (NADPH) to bovine liver glutamate dehydrogenase is controlled by the presence of phosphate, acetate, and other anions as well as the pH of the medium. These effectors mediate this binding by lowering the pK (8.5) of an ionizable group on the enzyme, and this pK shift is linked to a high enthalpy E <--> E' transition in the protein. In this study, we have measured enthalpy changes and proton transfer for enzyme-NADPH binding under a variety of combinations of phosphate, acetate, and hydrogen in the pairs acetate-NADPH and H(+)-phosphate, and negative interactions are seen in the pairs H(+)-NADPH, phosphate-NADPH, acetate-phosphate, and H(+)-acetate. We present a general model to account for all of these effects. This model incorporates a newly defined coenzyme binding subsite. The observed phenomena are interpreted in terms of the extent of loading of the specific anion-binding site on the enzyme that regulates the ionization of an enzyme group of pK 8.5. A proton is cooperatively shared with two phosphate groups at this site. Furthermore, we conclude that this cooperative trimolecular binding to the enzyme constitutes an allosteric driving force for the high enthalpy two-state transition observed in the ligand binding reactions of this enzyme.