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首页> 外文期刊>Biochemistry >1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase.
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1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase.

机译：1-甲基-4-苯基-1,2,3,6-四氢吡啶作为细胞色素P450 2D6的底物：NADPH-细胞色素P450还原酶的变构作用。

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摘要

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces Parkinsonism symptoms in man, has been examined as a substrate of recombinant human cytochrome P450 2D6. When cumene hydroperoxide is used as an oxygen and electron donor, a single product is formed, identified as 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) for formation of this product (130 microM) is in agreement with the dissociation constants for MPTP binding to the enzyme determined by optical and nuclear magnetic resonance (NMR) spectroscopy. When the reaction is carried out with nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) and recombinant human NADPH-cytochrome P450 reductase, a second product, identified as 1-methyl-4-(4'-hydroxyphenyl)-1,2,3,6-tetrahydropyridine, is formed in addition to 4-phenyl-1,2,3,6-tetrahydropyridine. The K(m) values for formation of these two products are 19 microM and 120 microM, respectively. Paramagnetic relaxation experiments have been used to measure distances betweenthe protons of bound MPTP and the heme iron, and these have been used to construct models for the position and orientation of MPTP in the active site. For the cytochrome alone, a single mode of binding was observed, with the N-methyl close to the heme iron in a position appropriate for the observed N-demethylation reaction. In the presence of the reductase, the data were not consistent with a single mode of binding but could be explained by the existence of two alternative orientations of MPTP in the active site. One of these, characterized by a dissociation constant of 150 microM, is essentially identical to that observed in the absence of the reductase. In the second, which has a K(d) of 25 microM, the MPTP is oriented so that the aromatic ring is close to the heme iron, in a position appropriate for p-hydroxylation leading to the formation of the product seen only in the presence of the reductase. In the case of codeine, another substrate for cytochrome P450 2D6, the addition of reductase had no effect on the nature of the product formed, the dissociation constant, or the orientation in the binding site. These observations show that NADPH-cytochrome P450 reductase has an allosteric effect on the active site of cytochrome P450 2D6 that affects the binding of some substrates but not others.
机译：1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）是一种在人体内产生帕金森氏症症状的神经毒素，已被检测为重组人细胞色素P450 2D6的底物。当异丙苯氢过氧化物用作氧和电子供体时，会形成单一产物，称为4-苯基-1,2,3,6-四氢吡啶。形成该产物的K（m）（130 microM）与通过光学和核磁共振（NMR）光谱测定的MPTP与酶的解离常数相符。当使用烟酰胺腺嘌呤二核苷酸磷酸（还原）（NADPH）和重组人NADPH-细胞色素P450还原酶进行反应时，第二种产物被鉴定为1-甲基-4-（4'-羟基苯基）-1,2,3除4-苯基-1，2，3，6-四氢吡啶外，还形成1，6-四氢吡啶。形成这两种产物的K（m）值分别为19 microM和120 microM。顺磁弛豫实验已用于测量结合的MPTP的质子与血红素铁之间的距离，并且已用于构建MPTP在活性部位的位置和方向的模型。对于单独的细胞色素，观察到单一结合模式，其中N-甲基接近血红素铁，处于适合于所观察到的N-脱甲基化反应的位置。在存在还原酶的情况下，数据与单一结合模式不一致，但可以通过活性位点中MPTP的两个替代方向的存在来解释。其中之一的特征在于解离常数为150 microM，与在没有还原酶的条件下观察到的基本上相同。在第二种中，其K（d）为25 microM，其MPTP的取向应使芳香环靠近血红素铁，处于适合于对羟基化作用的位置，导致仅在环戊烷中形成产物。还原酶的存在。在可待因的情况下，可待因是细胞色素P450 2D6的另一种底物，添加还原酶对形成的产物的性质，解离常数或结合位点的方向没有影响。这些观察结果表明，NADPH-细胞色素P450还原酶对细胞色素P450 2D6的活性位点具有变构作用，从而影响某些底物的结合，但不影响其他底物的结合。

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