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 modification of histidine residues of glutamate dehydrogenase (GDH) isoproteins from bovine brains with diethyl pyrocarbonate (DEPC). This suggests that the DEPC modified histidine residues are not critically involved in the catalysis of the GDH isoproteins. The influence of DEPC modified histidine residue(s) on binding of GTP to GDH isoproteins was investigated by protection studies. These studies showed that inhibition of GDH isoproteins by GTP was protected by preincubation of GDH isoproteins with DEPC. The amount of protection was dependent on the concentration of DEPC. The GTP inhibition was fully protected by preincubation of GDH isoproteins with DEPC at saturating concentrations. These results indicate that the histidine residues may play an important role in the GTP binding on GDH isoproteins. Spectrophotometric studies showed that three histidine residues per enzyme subunit were able 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 involved in the GTP binding domain of GDH isoproteins. The quantitative affinity 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 allosteric regulator, showed no significant changes in the interaction of DEPC with GDH isoproteins.
Lee
J
J
Department of Biochemistry, University of Ulsan College of Medicine, Seoul, Korea.
Lee
J E
JE
Cho
E H
EH
Choi
S Y
SY
Cho
S W
SW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D007527:Isoenzymes; D006639:Histidine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase; D019204:GTP-Binding Proteins; D004047:Diethyl Pyrocarbonate