Cnubben N H, Peelen S, Borst J W, Vervoort J, Veeger C, Rietjens I M
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Chem Res Toxicol. 1994 Sep-Oct;7(5):590-8. doi: 10.1021/tx00041a002.
The cytochrome P450 (P450) catalyzed 4-hydroxylation of halogenated anilines was investigated with special emphasis on possible relationships between kinetic parameters and physicochemical and electronic characteristics of the substrates. The most important observation of the present study was a correlation (r = 0.96) between the natural logarithm of the apparent maximum reaction rate kcats for 4-hydroxylation of the aniline substrates in a iodosobenzene-supported microsomal cytochrome P450-catalyzed reaction and the energy of the highest molecular orbital [E(HOMO)] of the anilines. This result is in accordance with a mechanism that proceeds by an initial electrophilic attack of the P450 (FeO)3+ intermediate on the frontier pi electrons of the aniline substrates. In the iodosobenzene-supported aniline 4-hydroxylation this electrophilic attack is the rate-limiting step. In the NADPH/oxygen-supported cytochrome P450-catalyzed 4-hydroxylation of the anilines a correlation of the natural logarithm of kcats with E(HOMO) was not observed and the kcats values were lower than observed in the iodosobenzene-supported reaction. From this result it is concluded that, although the NADPH/oxygen-supported microsomal 4-hydroxylation of the halogenated anilines proceeds by the same cytochrome P450 (FeO)3+ intermediate and, thus, by a similar electrophilic attack of the (FeO)3+ on the pi electrons of the substrate, this attack is no longer the rate-limiting step of the reaction. Additional results of the present study demonstrate that the apparent Michaelis constant Kms of the NADPH/oxygen-supported 4-hydroxylation of the anilines decreases with increasing hydrophobicity of the aniline derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了细胞色素P450（P450）催化卤代苯胺的4-羟基化反应，特别关注动力学参数与底物物理化学和电子特性之间的可能关系。本研究最重要的观察结果是，在碘苯支持的微粒体细胞色素P450催化反应中，苯胺底物4-羟基化的表观最大反应速率kcats的自然对数与苯胺的最高分子轨道能量[E(HOMO)]之间存在相关性（r = ０.９６）。该结果与一种机制相符，即该反应通过P450（FeO）3+中间体对苯胺底物前沿π电子的初始亲电攻击进行。在碘苯支持的苯胺4-羟基化反应中，这种亲电攻击是限速步骤。在NADPH/氧气支持的细胞色素P450催化的苯胺4-羟基化反应中，未观察到kcats的自然对数与E(HOMO)的相关性，且kcats值低于碘苯支持反应中的值。由此得出结论，尽管卤代苯胺的NADPH/氧气支持的微粒体4-羟基化反应通过相同的细胞色素P450（FeO）3+中间体进行，因此（FeO）3+对底物π电子的亲电攻击类似，但这种攻击不再是反应的限速步骤。本研究的其他结果表明，苯胺的NADPH/氧气支持的4-羟基化反应的表观米氏常数Kms随着苯胺衍生物疏水性的增加而降低。（摘要截短于250字）
