title: pH-dependent thermodynamic parameters of the glutamate dehydrogenase-alpha-ketoglutarate-NADPH complex.
abstract: The enthalpy of formation of the reactive bovine glutamate dehydrogenase-alpha-ketoglutarate-NADPH complex has been measured as a function of pH, at two temperatures and in two buffer systems having different enthalpies of ionization. The results demonstrate the existence of an extensive two-way traffic of protons between the buffer and the complex itself. While the pattern in a single buffer is too complex to resolve within the experimental limitations imposed by the system, we develop a theory which shows that the difference in delta H0H+ between buffers of different enthalpies of ionization has a simpler, more easily resolvable form. From such a relationship we show that in the process of binding of a molecule of NADPH to the enzyme-alpha-ketoglutarate complex, a proton has been shifted from the buffer to the complex and that this is due to the shift of the pK of an ionized group on the enzyme to a much higher value when the complex is formed. The delta Cp of formation of the complex is -450 cal . deg.-1 . M-1, and is pH independent. Implications of these results for the interpretation of enthalpies of enzyme-complex formation obtained at a single pH, and at one temperature, with one buffer system are mentioned. The complexity of the pattern of the dependence of delta H0, taken together with the magnitudes of the individual features of these dependencies, suggest that a number of large pK shifts occur when the enzyme-NADPH-alpha-ketoglutarate complex is formed. It is quite probable that these shifts represent a sizeable fraction of the machinery of the catalytic mechanism.
mesh_terms: D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D007656:Ketoglutaric Acids; D009249:NADP; D005969:Glutamate Dehydrogenase