title: NADPH binding induced proton ionization as a cause of nonlinear heat capacity changes in glutamate dehydrogenase.
abstract: Functional group interactions involved in the formation of the glutamate dehydrogenase-NADPH binary complex have been studied by three independent but complementary approaches: the pH dependence of the overall dissociation constant measured by an improved differential spectroscopic technique; the pH dependence of the enthalpy of complex formation measured by flow calorimetry; and the pH dependence of the number of protons released to, or taken up from, the solvent in the complex formation reaction, measured by titration. We conclude that the coenzyme binds to the enzyme through three distinguishable interactions: a pH-independent process involving the binding of the reduced nicotinamide ring; a relatively weak "proton-stabilizing" process, occurring at low pH involving the shift at a pK of 6.3 in the free enzyme to 7.0 in the enzyme-NADPH complex; and a stronger "proton-destabilizing" process, occurring at a higher pH involving a shift of a pK of 8.5 in the enzyme down to 6.9 in the enzyme-NADPH complex. The proton ionization of the free enzyme involved in this third interaction exhibits some unusual thermodynamic parameters, having delta Go = +11.5 +/- 0.1 kcal mol-1, delta Ho = +19 +/- 1 kcal mol-1, and delta So = +23 eu. We show here that this proton ionization step is directly related to and indeed constitutes the "implicit" shift in enzyme macrostates which we have shown to be responsible for the existence of large highly nonlinear delta Cpo effects in the formation of this complex [Fisher, H. F., Colen, A. H., & Medary, R. T. (1981) Nature (London) 292, 271-272].
mesh_terms: D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D008954:Models, Biological; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
chemical_list: D009249:NADP; D005969:Glutamate Dehydrogenase