The Mechanism of Ligand-induced Structural Changes in Glutamate Dehydrogenase

Changes in the weight average molecular weight and the spectrum of the reduced coenzyme subsequent to addition of coenzyme and guanine nucleotides to bovine liver glutamate dehydrogenase have been measured using stopped flow techniques. The data are consistent with an ordered sequence of events with respect to ligand-induced depolymerization of the enzyme. A mechanism is proposed for the ligand-induced effects which assumes that the ligand binding steps are in rapid equilibrium and that after ligands bind to the enzyme there are at least two conformational changes with the one induced by the guanine nucleotide occurring after that induced by the coenzyme. When DPNH is used as coenzyme, an additional, relatively slow conformational change is observed as a consequence of DPNH binding to a second, presumably nonactive site. A computer data fitting program is described which, using the assumed mechanism, allows determination of the apparent rate constants for these processes for each of the four pairs of nucleotides (TPNH-GTP, TPNH-GDP, DPNH-GTP, and DPNH-GDP). It is shown that each pair of nucleotides is associated with a different set of rate constants and, as a consequence, yields a rather different time and nucleotide concentration dependence of isomerization and depolymerization of the polymeric enzyme. Of most complexity are the results observed for the DPNH-GDP pair, in which depolymerization is clearly biphasic. For this case, the extent of one phase relative to the other is dependent on enzyme concentration. Only half-saturation with coenzyme (0.5 mole per mole of polypeptide chain) is required to give complete depolymerization in the presence of saturating GTP concentrations. There is no evidence for cooperative interactions between sites on the six polypeptide chains of the monomer.