Vatsis K P, Oprian D D, Coon M J
Acta Biol Med Ger. 1979;38(2-3):459-73.
Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.
进行了停流研究，以考察在25℃厌氧CO环境下，在存在NADPH-细胞色素P-450还原酶和磷脂的情况下，NADPH对5,6-苯并黄酮诱导的P-450 LM4的还原动力学。无论NADPH浓度或还原酶与P-450 LM4的摩尔比如何，该反应均呈现双相动力学。快速相和慢速相的表观一级速率常数分别确定为0.9至1.0和0.25 s-1。当还原酶和P-450 LM4以等摩尔量存在时，被还原的P-450 LM4总量随NADPH浓度呈线性增加；滴定得到的化学计量比为每摩尔还原酶-细胞色素复合物2摩尔NADPH。NADPH浓度对快速相和慢速相的一级速率常数大小没有明显影响。在存在苄非他明的情况下获得的动力学与不存在该底物时观察到的动力学基本无法区分，而当从反应混合物中省略磷脂时，快速相中被还原的P-450 LM4量减少，但该相的速率常数不变。当还原酶与P-450 LM的摩尔比为1.0时，可获得P-450 LM2或LM4对NADPH的近最大氧化速率。苄非他明增强了P-450 LM2对NADPH的氧化，但对P-450 LM4的活性没有影响。当从重组酶系统中省略磷脂时，存在P-450 LM2和LM4时NADPH的氧化速率分别降低了80%和40%。这些研究为还原酶与P-450 LM4之间形成催化功能的1:1复合物提供了证据，并表明P-450 LM2和LM4在对磷脂的依赖性方面存在差异。
