Studies on the cosubstrate site of protease solubilized NADPH-cytochrome P450 reductase.
Studies on the cosubstrate site of protease solubilized NADPH-cytochrome P450 reductase.
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蛋白酶溶解 NADPH-细胞色素 P450 还原酶共底物位点的研究。
DOI：
10.1111/j.1399-3011.1980.tb02940.x
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
T. Lazar
中科院分区：
文献类型：
--
作者：
L. Lumper;F. Busch;S. Dzelić;J. Henning;T. Lazar
修改的蛋白酶溶解的NADPH-细胞色素P450还原酶（= NADPH-细胞色素c还原酶）在关键的SH基团的共底物结合位点的影响KmNADPH，但不是V的细胞色素c还原。KmNADPH的增加取决于引入的取代基的大小和电荷。用CN-、S_2O_3-和（N-乙基）琥珀酰亚胺基取代共底物位点SH分别使KmNADPH增加3倍、7倍和23倍。在1 mM NADP+存在下，还原酶与未标记NEM预孵育后，NADPH结合区中的关键SH基团可通过N-乙基（2，3 - 14 C）马来酰亚胺特异性放射性标记。通过对还原酶的溴化氰片段进行肽图分析和尿素SDS凝胶电泳，证明了在共底物位点上的必需半胱氨酸的选择性反应。蛋白酶溶解的NADPH-细胞色素P450还原酶被针对组氨酸、精氨酸和赖氨酸残基的试剂灭活。NADP（H）（1 mM）和2 '-AMP（1 mM）仅对1，2-环己二酮（12 mM）的反应提供有效保护。因此，通过所述的修饰实验不能确定碱性氨基酸残基对NADPH-细胞色素P450还原酶的共底物结合的功能作用。NADPH-细胞色素P450还原酶中NADPH结合位点的数量通过用CD-光谱法监测的NADP+滴定酶确定为1个位点/mol还原酶。
Modification of the protease solubilized NADPH-cytochrome P450 reductase (= NADPH-cytochrome c reductase) at the critical SH group in the cosubstrate binding site affects KmNADPH but not V for the cytochrome c reduction. The increase of KmNADPH is dependent on the size and the charge of the substituent introduced. Substitution of the cosubstrate site SH by the CN-, S2O3- and the (N-ethyl) succinimido group effects a 3-, 7- and 23-fold increase of KmNADPH, respectively. The critical SH group in the NADPH binding region can be specifically radiolabeled by N-ethyl (2,3-14C) maleimide after preincubation of the reductase with unlabeled NEM in the presence of 1 mM NADP+. The selective reaction at the essential cysteine in the cosubstrate site is demonstrated by peptide mapping of the thermolytic digest and urea SDS gel electrophoresis of the cyanogen bromide fragments of the reductase. Protease solubilized NADPH-cytochrome P450 reductase is inactivated by reagents directed to histidine, arginine and lysine residues. NADP (H) (1 mM) and 2'-AMP (1 mM) give effective protection only for the reaction of 1,2-cyclohexanedione (12 mM). The functional role of the basic amino acid residues for the cosubstrate binding by the NADPH-cytochrome P450 reductase cannot be established therefore by the modification experiments described. The number of NADPH binding sites in the NADPH-cytochrome P450 reductase is determined to one site/mol reductase by titration of the enzyme with NADP+ monitored by CD-spectroscopy.