KINETICS OF ALCOHOL METABOLIZING ENZYMES

这是在申请ADAMHA科学家发展奖。 很长的- 本研究项目的长期目标是分析动力学 酒精代谢酶的特性。 假设人类 醇脱氢酶的动力学机制和限速步骤不同， 慢性酒精消耗扰乱了 微粒体乙醇氧化系统（MEOS）。 摄入的酒精 主要由醇脱氢酶或MEOS代谢。 在 至少五种醇脱氢酶亚基在肝脏中表达， 包括alpha、beta和gamma（I类）、eta（II类）和chi（II类 III）。 第六类醇脱氢酶，sigma，表达于 胃，并可能参与首过代谢。 这些同工酶， 在底物结合位点48和93处具有天然取代， 位点，在底物特异性上有很大不同。 天然替代物 β亚基中His-47或Cys-369对Arg-47和Arg-369的取代产生 同工酶在辅酶结合亲和力上有显著差异。 MEOS 是一种含有P450同工酶的膜结合多酶复合物。 长- 大鼠长期酒精暴露导致肝脏乙醇含量增加， 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。 我已经通过停流技术检查了辅酶结合 人β 1 β 1酶在E.杆菌 47位的突变。 我将利用这个科学家发展奖， 开发停流法检测辅酶和底物 表观结合速率常数和极限氢化物转移速率 在位置369处含有位点特异性突变的β 1 β 1酶，和 胃中的σ-乙醇脱氢酶 我会发展研究技术 利用停流动力学研究动力学机理， P450 IIE的底物特异性，并确定长- 长期酒精暴露对微粒体脂质体中P450 IIE机制影响 我 还将使用定点突变和分子图形来表达 并纯化在位置48和93处具有位点特异性突变的酶， 然后用这些突变体构建基质的晶格结构， 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.