Single turnover studies with oxy-cytochrome P-450cam.
Single turnover studies with oxy-cytochrome P-450cam.
复制标题
使用氧细胞色素 P-450cam 进行单周转研究。
DOI：
10.1016/0003-9861(86)90029-9
复制
发表时间：
1986
影响因子：
3.9
通讯作者：
Peterson,JA
中科院分区：
文献类型：
--
作者：
Brewer,CB;Peterson,JA
本文用停流分光光度法研究了细菌细胞色素P-450 cam的催化步骤，即反应循环中产物5-外羟基樟脑的形成和释放。我们的方法是观察还原的恶臭还蛋白和氧合的樟脑结合的细胞色素P-450cam之间的单周转反应。在产品释放后，使用抑制剂美泰龙捕获细胞色素，从而防止另一种樟脑分子的结合，从而防止多次翻转。在几个波长测量了该反应的时间进程，并发现该反应是两相的。反应的第二阶段相对缓慢，是铁还原，甲孕酮结合的细胞色素P-450钙调素。第一阶段与气相色谱测定的细胞色素P-450 cam的化学计量形成相一致。第一阶段的详细动力学研究表明，在固定的细胞色素P-450 cam的极限浓度下，初始速率与putidaredoin浓度呈双曲线依赖关系。细胞色素的Vmax为每秒53μm/μ，恶臭还蛋白的Km为33μm，初始速率与恶臭还蛋白浓度之间的双曲线关系支持细胞色素快速与恶臭还蛋白结合，然后经历一个或多个较慢的复合体内步骤。
The catalytic step of bacterial cytochromeP-450cam, i.e., the step of the reaction cycle in which the product 5-exo-hydroxycamphor is formed and released by the enzyme, has been studied by stopped-flow spectrophotometry. Our approach has been to observe a single-turnover reaction between reduced putidaredoxin and oxygenated camphor-bound cytochromeP-450cam. Multiple turnovers are prevented by using the inhibitor metyrapone to trap the cytochrome after product release, which prevents binding of another camphor molecule. The time course of the reaction has been measured at several wavelengths and has been found to be biphasic. The relatively slow second phase of the reaction is the reduction of ferric, metyrapone-bound cytochromeP-450cam. The first phase coincides with the formation of product stoichiometrically with cytochromeP-450cam, as measured by gas chromatography. A detailed kinetic study of the first phase reveals a hyperbolic dependence of initial rate upon putidaredoxin concentration at a fixed, limiting concentration of cytochromeP-450cam. TheVmaxis 53 μmper second per μmcytochrome, and theKmfor putidaredoxin is 33 μm. The hyperbolic relationship between initial rate and putidaredoxin concentration supports a model in which the cytochrome rapidly binds putidaredoxin, then undergoes one or more slower intracomplex steps.