REGULATION OF MICROBIAL OXIDATION REACTIONS

这项研究计划的长期目标是达成一项理解 细胞色素P-450的催化机制及其调控 单氧合反应。这些酶很重要，因为它们 参与药物的氧化，类固醇激素的合成， 致癌物质的激活和多不饱和脂肪酸的代谢。 研究的重点是细菌细胞色素P-450，它催化 樟脑的氧化，因为这个酶系统很容易分离 纯形式的，数量足以用于本研究。 在即将到来的赠款期间，研究将集中在 确定在反应过程中形成的瞬时中间产物 还原型恶臭还蛋白对细胞色素P-450的还原作用 还原剂。这些中间体的形成和衰变的动力学将 采用停流-温度跳跃分光光度法进行测定。这个 中间体将通过冷冻淬火技术被捕获，从而使EPR 可以检查光谱并确认中间体的身份。 细胞色素P-450总反应的动力学和控制 用适当的计算机模型进行评估，以便精确控制 可以更好地理解这一重要的酶系统。

The long term goal of this research program is to attain an understanding of the mechanism and regulation of cytochrome P-450 catalyzed monooxygenation reactions. These enzymes are important because they are involved in the oxidation of drugs, synthesis of steroid hormones, activation of carcinogens and metabolism of polyunsaturated fatty acids. The research has focused on the bacterial cytochrome P-450 which catalyzes the oxidation of camphor because this enzyme system can be readily isolated in pure form in quantities which are sufficient to be used for this study. In the coming grant period the research will be focused on the identification of the transient intermediates which are formed during the reduction of cytochrome P-450 by reduced putidaredoxin, the physiological reductant. The kinetics of formation and decay of these intermediates will be measured with stopped-flow and temperature jump spectrophotometry. The intermediates will be trapped by freeze-quench techniques so that the EPR spectrum can be examined and the identity of the intermediates confirmed. The kinetics and control of the overall reaction of cytochrome P-450 will be evaluated with an appropriate computer model so that the precise control of this important enzyme system can be better appreciated.