MECHANISM OF GLYOXALASE I AND FORMALDEHYDE DEHYDROGENASE

乙二酸酶I和甲醛脱氢酶(FDH)都是谷胱甘肽 (GSH)依赖的酶，催化各种醛的转化 到谷胱甘肽的硫酯加合物。这两种酶都必须在 相互转换的非对映异构体的混合物，形成于 谷胱甘肽硫原子与羰基之间的预平衡步骤 醛的基团。乙草酸酶I可以非立体特异性地转化 这两种非对映异构体直接转化为产物是一种新颖的产物 本实验室发展的同位素捕捉法。整体而言 拟议研究的目标是确定催化剂 这一观察的意义和分子基础以及检验 甲醛脱氢酶的性质是否与 乙草酸酶I.为实现这一目标，进行了以下实验 建议：首先，将FDH的底物立体特异性 由同位素捕捉法建立。第二，评估 假设非立体特异性底物的“催化优势” 结合是指酶催化不受非酶速率的限制。 半巯基非对映异构体的相互转化，这些非酶速率 将作为条件的函数，通过核磁共振展宽来确定 方法：研究方法。第三，作为乙草酸酶I和FDH催化 结合的非对映异构体的异构化作为一种对等反应，该酶 亚甲基质子共振的诱导核磁共振谱线展宽 将对非对映体进行检测。最后，假设能力 乙醛酸酶I容纳两个非对映异构体作为底物的原因是 键合的谷胱甘肽基硫原子的位置迁移率 非对映异构体将从结合构象间接评估 同分异构体类似于Mn++乙二醛酶I的底物，由 通过T1上的顺磁效应测量的Mn++到质子的距离。

Glyoxalase I and formaldehyde dehydrogenase (FDH) are both glutathione (GSH)-dependent enzymes that catalyze the conversion of various aldehydes to thioester adducts of glutathione. Both enzymes must operate on a mixture of interconverting diasteriomeric hemimercaptals, formed in a preequilibrium step between the sulfur atom of glutathione and the carbonyl group of the aldehyde. That glyoxalase I can nonstereospecifically convert both diasteriomers directly to product is an outcome of a novel isotope-trapping method developed in this laboratory. The overall objective of the proposed research is to determine the catalytic significance and molecular basis of this observation as well as to test whether formaldehyde dehydrogenase has similar properties in common with glyoxalase I. To achieve this objective the following experiments are proposed: First, the substrate stereospecificity of FDH will be established by isotope trapping methods. Second, to evaluate the hypothesis that the "catalytic advantage" of nonstereospecific substrate binding is that enzymic catalysis is not limited by the nonenzymic rates of interconversion of the hemimercaptal diasteriomers, these nonenzymic rates will be determined, as a function of conditions, by nmr-broadening methods. Third, as a test that glyoxalase I and FDH catalyze the ipimerization of the bound diasteriomers as a parital reaction, the enzyme induced nmr line-broadening of the methine proton resonances of the diasteriomers will be tested for. Finally, the hypothesis that the ability of glyoxalase I to accomodate both diasteriomers as substrates is due to positional mobility of the glutathionyl sulfur atom of the bound diasteriomers will be indirectly evaluated from the binding conformation of isomeric substrate analogs to Mn++ glyoxalase I, as determined from the Mn++ to proton distances measured by paramagnetic effects on T1.