Identification of ADP-ribosylation site in human glutamate dehydrogenase isozymes.
When the influence of ADP-ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 microM NAD+ for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP-ribosylation in mitochondria. ADP-ribosylated hDGH isozymes were reactivated by Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase. The stoichiometry between incorporated ADP-ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP-ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP-ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP-ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP-ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP-ribosylation.
Choi
Myung-Min
MM
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, 388-1 Poongnap-2dong, Songpa-gu, Seoul 138-736, South Korea.
Huh
Jae-Wan
JW
Yang
Seung-Ju
SJ
Cho
Eun Hee
EH
Choi
Soo Young
SY
Cho
Sung-Woo
SW
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics
D007527:Isoenzymes; D000246:Adenosine Diphosphate Ribose; D005969:Glutamate Dehydrogenase