KINETICS OF ALCOHOL METABOLIZING ENZYMES

这是在申请ADAMHA科学家发展奖。长的- 本研究项目的学期目标是分析动力学 酒精代谢酶的性质。假设是人类 乙醇脱氢酶在动力学机制和限速步骤上有所不同， 而长期饮酒扰乱了 微粒体醇氧化系统(MEOS)。摄入的酒精是 主要由酒精脱氢酶或meos代谢。在… 至少有五个酒精脱氢酶亚基在肝脏和 包括阿尔法、贝塔和伽马(I类)、ETA(II类)和CHI(类 三)。第六类酒精脱氢酶，西格玛，表达在 胃，并可能参与首过代谢。这些同工酶， 在底物结合中的第48位和第93位具有自然取代 在底物专一性上有很大差异。自然替代 His-47或Cys-369中的Arg-47和β亚基中的Arg-369产生 同工酶在辅酶结合亲和力上有很大不同。梅奥斯 是一种膜结合的多酶复合体，含有P450同工酶。长- 长期酒精暴露导致大鼠肝脏乙醇含量增加- 诱导同工酶P450 IIE，并改变细胞膜组成。 这些变化可能影响P450 IIE同工酶的动力学机制。 我用停流技术检测了辅酶结合 人β1β1酶在大肠杆菌中的表达及其特性 47位发生突变。我将利用这个科学家发展奖来 建立检测辅酶和底物的停流法 表观结合速率常数和极限氢化物转移速率 在第369位和第369位含有定点突变的Beta1beta1酶 胃Sigma-酒精脱氢酶。我将开发研究技术 为了用停流动力学来研究其动力学机制， P450 IIE的底物专一性，并确定Long-Long-DNA的作用。 长期酒精暴露对微粒体脂质体中P450 IIE机制的影响。我 还将使用定点突变和分子图形来表达 并通过第48和93位的定点突变来提纯酶，以及 然后用这些突变体构建底物的晶格结构 分子图形的特异性。

This is in application for the ADAMHA Scientist Develop Award. The long- term objective of this research project is to analyze the kinetic properties of alcohol-metabolizing enzymes. The hypothesis is that human alcohol dehydrogenases vary in kinetic mechanism and rate-limiting step, and that chronic alcohol consumption perturbs the kinetic mechanism of the microsomal ethanol-oxidizing system (MEOS). Ingested alcohols are metabolized predominantly by either alcohol dehydrogenases or MEOS. At least five alcohol dehydrogenase subunits are expressed in the liver and include alpha, beta, and gamma (Class I), eta (Class II), and chi (Class III). A sixth class of alcohol dehydrogenase, sigma, is expressed in the stomach and may be involved in first-pass metabolism. These isoenzymes, with natural substitutions at positions 48 and 93 in the substrate binding site, differ considerably in substrate specificity. Natural substitutions of His-47 or Cys-369 for Arg-47 and Arg-369 in the beta subunit produces isoenzymes that differ dramatically in coenzyme binding affinity. The MEOS is a membrane-bound multi-enzyme complex containing P450 isoenzymes. Long- term alcohol exposure in rats leads to an increase in a hepatic ethanol- induced isoenzyme, P450 IIE, and alters membrane composition in the cells. These changes may affect the kinetic mechanism of the P450 IIE isoenzyme. I have examined by stopped-flow techniques the coenzyme binding characteristics of human beta1beta1 enzymes expressed in E. coli with mutations at position 47. I will use this Scientist Development Award to develop stopped-flow methods for examining the coenzyme and substrate apparent binding rate constants and limiting hydride transfer rates of beta1beta1 enzymes containing site-specific mutations at position 369 and of stomach sigma-alcohol dehydrogenase. I will develop research techniques for using stopped-flow kinetics to study the kinetic mechanism and substrate specificity of P450 IIE, and to determine the effects of long- term alcohol exposure on P450 IIE mechanism in microsomal liposomes. I will also use site-directed mutagenesis and molecular graphics to express and purify enzymes with site-specific mutations at positions 48 and 93, and then with these mutants construct a lattice structure of substrate specificity with molecular graphics.