MECHANISM OF GLYOXALASE I AND FORMALDEHYDE DEHYDROGENASE

谷胱甘肽酶I和甲醛脱氢酶（FDH）都是谷胱甘肽 催化各种醛转化的（GSH）依赖性酶 谷胱甘肽的硫酯加合物。 这两种酶都必须在 相互转化的双异构半硫醇的混合物，在 谷胱甘肽的硫原子和羰基之间的预平衡步骤 醛的基团。 我可以将这种糖苷酶非立体特异性地转化为 两种异构体直接转化为产物是一种新颖的产物， 本实验室开发的同位素捕获方法。 整体 该研究的目的是确定催化剂 这一观察的意义和分子基础以及测试 甲醛脱氢酶是否具有与 谷胱甘肽酶I 为了实现这一目标，进行了以下实验： 建议：首先，FDH的底物立体特异性将是 通过同位素捕获方法建立的。 第二，评价 假设非立体特异性底物的“催化优势” 结合是酶催化不受非酶速率的限制， 半巯基二聚体的相互转化，这些非酶速率 作为条件的函数， 方法. 第三，作为glycoprotein酶I和FDH催化蛋白质水解的试验， 结合的二聚体的异构化作为一个部分反应，酶 的次甲基质子共振的诱导NMR谱线增宽 将测试双链异构体。 最后，假设 的glycoprotein酶I，以容纳作为底物的两个双异构体是由于 键合的谷胱甘肽硫原子的位置迁移率 将从以下物质的结合构象间接评价双链异构体： Mn++糖苷酶I的异构底物类似物，如从 Mn++质子距离测量顺磁效应T1。

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.