Changes in preferential hydration of glyceraldehyde 3-phosphate dehydrogenase from yeast brought about by coenzyme binding;
The binding of nicotinamide-adenine dinucleotide to glyceraldehyde 3-phosphate dehydrogenase from yeast was investigated over a temperature and pH range by methods of fluorescence quenching, sedimentation equilibrium, and nuclear magnetic relaxation in order to characterized aspects of the protein conformational change, and concomitant change is salvation, that are associated with coenzyme binding. Binding equilibria were measured by the fluorescence quenching method. The isotherms were sigmoidal in shape at pH 8.5 and hyperbolic at pH 7.4 and 6.5. The apparent binding constants varied inversely with temperature and stoichiometry was four binding sites per molecule to tetrameric enzyme. Scatchard plots of the data indicated that the dehydrogenase displays positive cooperativity in coenzyme binding at pH 8.5, negative cooperativity in binding at pH 6.5, and that the binding sites were equivalent and independent at pH 7.4. These results are in accord with and amplify previously published data, obtained under a more restricted range of conditions. Buoyant densities of the enzyme and its coenzyme complex were measured (a) by analytical equilibrium sedimentation in cesium chloride gradients and (b) by extrapolating the product of sedimentation coefficient and solvent viscosity to zero as a function of solvent density in a series of potassium phosphate solutions. Formation of the NAD complex in both cases resulted n a increase in buoyant density associated with a significant decrease in preferential hydration, the magnitude of which is a function of temperature and the nature of the salt. In the buoyant density measurements in cesium chloride, the associated changed in the binding of chloride ions were also measured. The results of the study are in agreement with independent typed of evidence that NAD addition to the enzyme results in a contraction of the quatemary structure of the protein. The water proton spin lattice relaxation rate, T1-1 measured at 3.5 MHz in a pulsed nuclear magnetic spectrometer in enhanced in the presence of the dissolved protein. A further enhancement, following the course of a binding isotherm, occurred on formation of the coenzyme complex. If the NAD effect were the result of dehydration, there would have been an increase rather than a decrease in the relaxation times. The observed effect on the water proton T1-1 is thus attributed to a restricted small class of bound water molecules that lose degrees of freedom as a result of the protein conformational transition. Such a result is in accord with theory but not previously been observed experimentally.