Orellano E G, Vallejos R H, Cazzulo J J
Comp Biochem Physiol B. 1985;80(3):563-8. doi: 10.1016/0305-0491(85)90291-3.
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.
从克氏锥虫图拉韦恩株（Tul 2株）的无鞭毛体中纯化得到的烟酰胺腺嘌呤二核苷酸磷酸（NADP）连接的谷氨酸脱氢酶（NADP - gluDH），受到汽巴克隆蓝FG3A的抑制，并在与苯乙二醛或伍德沃德试剂K预孵育时失活。汽巴克隆蓝FG3A的抑制作用对NADPH具有竞争性，表观抑制常数Ki为20微摩尔，这表明该酶呈现出大多数脱氢酶和激酶所特有的“二核苷酸折叠”特征。用苯乙二醛预孵育使NADP - gluDH失活，反应级数为1，以及α - 酮戊二酸提供的部分保护作用，表明该酶活性位点存在一个精氨酸残基，它可能通过与底物的一个羧基相互作用参与α - 酮戊二酸的结合。用伍德沃德试剂K预孵育使NADP - gluDH失活，表明活性位点存在一个来自天冬氨酸或谷氨酸残基的羧基，它可能参与阳离子底物NH₄⁺的结合。在与该试剂预孵育期间存在NADPH会增加失活速率，这表明辅酶的结合增加了反应性羧基的暴露。
