Shet M S, Faulkner K M, Holmans P L, Fisher C W, Estabrook R W
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038, USA.
Arch Biochem Biophys. 1995 Apr 20;318(2):314-21. doi: 10.1006/abbi.1995.1235.
The recombinant fusion protein containing the heme domain of human P450 3A4 and the flavin domains of rat NADPH-cytochrome P450 (P450) reductase (rF450[mHum3A4/mRatOR]L1) requires both phospholipid and detergent as well as cytochrome b5 (b5) for the NADPH-dependent catalysis of the 6 beta-hydroxylation of testosterone. NADPH oxidation results in the formation of hydrogen peroxide in the presence or absence of phospholipid and detergent. NADPH oxidation and hydrogen peroxide formation are inhibited by the addition of b5 and stimulated greater than 3-fold by the addition of testosterone. Marked differences in the ability of various phospholipids to support the P450-dependent 6 beta-hydroxylation of testosterone by the fusion protein were seen. Addition of a 4-fold excess of purified NADPH-P450 reductase, in the presence of phospholipid, detergent, and b5, stimulates the rate of testosterone 6 beta-hydroxylation approximately 10-fold, providing turnover rates as high as 80 min-1 for P450 3A4. Approximately 30% of the rate of hydrogen peroxide formation is not sensitive to inhibition by the P450 inhibitor ketoconazole, suggesting hydrogen peroxide (or superoxide anion) formation directly from the reduced flavin domains of the fusion protein. It is proposed that the stimulation of NADPH oxidation observed following the addition of testosterone to the fusion protein may serve as a useful means of monitoring the interaction of other substrates with this P450 and thereby permit the rapid screening of chemicals to evaluate their potential metabolism by a human P450.
包含人P450 3A4血红素结构域和大鼠NADPH - 细胞色素P450（P450）还原酶黄素结构域的重组融合蛋白（rF450[mHum3A4/mRatOR]L1）在NADPH依赖的睾酮6β-羟基化催化过程中，既需要磷脂和去污剂，也需要细胞色素b5（b5）。无论有无磷脂和去污剂，NADPH氧化都会导致过氧化氢的形成。添加b5可抑制NADPH氧化和过氧化氢形成，而添加睾酮则可使其刺激作用增强3倍以上。观察到各种磷脂在支持融合蛋白对P450依赖的睾酮6β-羟基化能力方面存在显著差异。在磷脂、去污剂和b5存在的情况下，添加4倍过量的纯化NADPH - P450还原酶，可使睾酮6β-羟基化速率提高约10倍，P450 3A4的周转速率高达80 min-1。约30%的过氧化氢形成速率对P450抑制剂酮康唑的抑制不敏感，这表明过氧化氢（或超氧阴离子）直接从融合蛋白的还原黄素结构域形成。有人提出，向融合蛋白中添加睾酮后观察到的NADPH氧化刺激作用，可能是监测其他底物与该P450相互作用的一种有用方法，从而能够快速筛选化学物质，以评估它们被人P450代谢的潜力。
