Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin.
Kinetic isotope effects on cytochrome P-450-catalyzed oxidation reactions. The oxidative O-dealkylation of 7-ethoxycoumarin.
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动力学同位素对细胞色素 P-450 催化氧化反应的影响。
DOI：
10.1016/s0021-9258(17)43248-0
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发表时间：
1984
期刊：
影响因子：
--
通讯作者：
A. Y. Lu
中科院分区：
文献类型：
--
作者：
G. Miwa;J. Walsh;A. Y. Lu
用两种纯化的细胞色素P-450同工酶催化7-乙氧基香豆素的O-脱乙基反应，测量了初级氚和氚同位素对Vm/Km和Vm的影响。根据这些数据，计算了本征同位素效应，如D.B.Northrop(生物化学(1975)14,2644-2651)所述。从苯巴比妥和3-甲基胆蒽诱导的大鼠肝脏分离的同工酶，观察到的氚同位素效应对Vm/Km的影响分别为3.79和1.90。然而，计算的本征同位素效应(KH/Kd=12.8到14.0)与观测到的两种酶的同位素效应对Vm/Km的影响相似，而且要大得多。这表明，本征同位素效应在同位素敏感的C-H键裂解步骤之前的各种步骤中被衰减，从而导致观察到的同位素效应的低值。因此，观测到的同位素效应不能准确反映与该反应有关的本征同位素效应的大小。在使用H_218O或18O_2的研究中，没有观察到18O掺入7-羟基香豆素产物中，表明酚氧完全来自底物。综上所述，这些数据提供了令人信服的证据，证明在半缩醛中间体的强制形成过程中，两种细胞色素P-450同工酶通过相同的自由基重组机制催化该底物的O-脱烷基反应。
The primary deuterium and tritium isotope effects on Vm/Km and on Vm have been measured for the O-deethylation of 7-ethoxycoumarin catalyzed by two purified isozymes of cytochrome P-450. From these data the intrinsic isotope effects have been calculated as described by D. B. Northrop (Biochemistry (1975) 14, 2644-2651). The observed deuterium isotope effects on Vm/Km are 3.79 and 1.90 for the isozymes isolated from the livers of rats induced by phenobarbital and 3-methylcholanthrene, respectively. The calculated intrinsic isotope effects, however, are similar and much larger (kH/kD = 12.8 to 14.0) than the observed isotope effects on Vm/Km for the two enzymes. This demonstrates that the intrinsic isotope effects are attenuated by various steps preceding the isotopically sensitive C-H bond cleavage step resulting in the low values for the observed isotope effects. Thus, the observed isotope effects do not accurately reflect the magnitude of the intrinsic isotope effect associated with this reaction. No incorporation of 18O into the 7-hydroxycoumarin product was observed in studies employing H218O or 18O2 demonstrating that the phenolic oxygen arises exclusively from the substrate. Taken together, these data provide compelling evidence that both cytochrome P-450 isozymes catalyze the O-dealkylation of this substrate by an identical radical recombination mechanism during the obligatory formation of a hemiacetal intermediate.