Huete-Pérez J A, Wu J C, Whitby F G, Wang C C
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Biochemistry. 1995 Oct 24;34(42):13889-94. doi: 10.1021/bi00042a021.
The IMP dehydrogenase from Tritrichomonas foetus has been identified as a potential target for antitritrichomonial chemotherapy. The gene encoding this enzyme was expressed in transformed Escherichia coli, and the recombinant protein was purified to homogeneity with an average yield of 3 mg of protein per liter of bacterial culture. Kinetic characterizations verified that the recombinant enzyme is in the authentic native state. 6-Cl-IMP, an irreversible inhibitor of the enzyme, was found to protect cysteine residue 319 of the enzyme against carboxymethylation by iodoacetamide. Radiolabeled IMP was covalently bound to the enzyme during the enzyme-catalyzed reaction via the formation of a specific adduct with cysteine residue 319. It is thus postulated that the conversion of IMP to XMP catalyzed by the IMP dehydrogenase from T. foetus is mediated by a nucleophilic attack of cysteine-319 in the enzyme protein to IMP at, most likely, its 2-position to facilitate a hydride transfer to NAD, resulting in the formation of a covalent intermediate between substrate and enzyme.
胎儿三毛滴虫的肌苷酸脱氢酶已被确定为抗三毛滴虫化疗的一个潜在靶点。编码该酶的基因在转化的大肠杆菌中表达，重组蛋白被纯化至均一，每升细菌培养物的平均蛋白产量为3毫克。动力学特性验证了重组酶处于真实的天然状态。发现该酶的不可逆抑制剂6-氯肌苷酸可保护酶的半胱氨酸残基319不被碘乙酰胺羧甲基化。在酶催化反应过程中，放射性标记的肌苷酸通过与半胱氨酸残基319形成特定加合物而与酶共价结合。因此推测，胎儿三毛滴虫的肌苷酸脱氢酶催化肌苷酸转化为黄苷酸是由酶蛋白中的半胱氨酸-319对肌苷酸最有可能在其2位进行亲核攻击介导的，以促进氢化物转移至烟酰胺腺嘌呤二核苷酸，从而在底物和酶之间形成共价中间体。
