Chemical modification of the active site of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi.
The NADP-linked glutamate dehydrogenase (NADP-gluDH) purified from epimastigotes of the Tulahuén strain, Tul 2 stock, of Trypanosoma cruzi, was inhibited by Cibacron Blue FG3A, and inactivated by preincubation with phenylglyoxal or Woodward's Reagent K. The inhibition by Cibracron Blue FG3A, competitive towards NADPH with an apparent Ki of 20 microM, suggests that the enzyme presents the "dinucleotide fold" characteristic of most dehydrogenases and kinases. The inactivation of the NADP-gluDH by preincubation with phenylglyoxal, with a reaction order of 1, and the partial protection afforded by alpha-oxoglutarate, suggest the presence of one arginine residue in the active site of the enzyme, which might participate in the binding of alpha-oxoglutarate through interaction with one of the carboxyl groups of the substrate. The inactivation of the NADP-gluDH by preincubation with Woodward's Reagent K suggests the presence of a carboxyl group, from an aspartic or glutamic acid residue, at the active site, which might participate in the binding of the cationic substrate NH+4. The presence of NADPH during preincubation with the reagent increased the inactivation rate, which suggests that binding of the coenzyme increases the exposure of the reactive carboxyl group.
Orellano
E G
EG
Vallejos
R H
RH
Cazzulo
J J
JJ
D000818:Animals; D001120:Arginine; D001665:Binding Sites; D001667:Binding, Competitive; D055598:Chemical Phenomena; D002621:Chemistry; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018086:Glutamate Dehydrogenase (NADP+); D007202:Indicators and Reagents; D007555:Isoxazoles / Q000494:pharmacology; D009249:NADP / Q000494:pharmacology*; D010658:Phenylglyoxal / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000201:enzymology*
D007202:Indicators and Reagents; D007555:Isoxazoles; D014227:Triazines; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D009249:NADP; C012842:Cibacron Blue F 3GA; D001120:Arginine; D005969:Glutamate Dehydrogenase; D018086:Glutamate Dehydrogenase (NADP+); D010658:Phenylglyoxal