An essential histidine residue in GTP binding domain of bovine brain glutamate dehydrogenase isoproteins

Greater than 90% of the original activity of the enzymes remained after mod ification of histidine residues of glutamate dehydrogenase (GDH) isoprotein s from bovine brains with diethyl pyrocarbonate (DEPC). This suggests that the DEPC modified histidine residues are not critically involved in the cat alysis of the GDH isoproteins. The influence of DEPC modified histidine res idue(s) on binding of GTP to GDH isoproteins was investigated by protection studies. These studies showed that inhibition of GDH isoproteins by GTP wa s protected by preincubation of GDH isoproteins with DEPC. The amount of pr otection was dependent on the concentration of DEPC. The GTP inhibition was fully protected by preincubation of GDH isoproteins with DEPC at saturatin g concentrations. These results indicate that the histidine residues may pl ay an important role in the GTP binding on GDH isoproteins. Spectrophotomet ric studies showed that three histidine residues per enzyme subunit were ab le to react with DEPC in the absence of GTP, whereas two histidine residues per enzyme subunit interacted with DEPC when the enzymes were preincubated with GTP. These results indicate that one of the histidine residues is inv olved in the GTP binding domain of GDH isoproteins. The quantitative affini ty chromatographic studies showed that the influence of GTP on the binding of GDH isoproteins to DEPC-Sepharose was significantly distinct for the two GDH isoproteins. GDH I was more sensitively affected by GTP than GDH II in the binding affinity for DEPC-Sepharose. ADP, another well-known allosteri c regulator, showed no significant changes in the interaction of DEPC with GDH isoproteins.