MOLECULAR ORBITAL-BASED QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP FOR THE CYTOCHROME P450-CATALYZED 4-HYDROXYLATION OF HALOGENATED ANILINES

The cytochrome P450 (P450) catalyzed 4-hydroxylation of halogenated an ilines was investigated with special emphasis on possible relationship s between kinetic parameters and physicochemical and electronic charac teristics of the substrates. The most important observation of the pre sent study was a correlation (r = 0.96) between the natural logarithm of the apparent maximum reaction rate k(cat)(S) for 4-hydroxylation of the aniline substrates in a iodosobenzene-supported microsomal cytoch rome P450-catalyzed reaction and the energy of the highest molecular o rbital [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 su bstrates. In the iodosobenzene-supported aniline 4-hydroxylation this electrophilic attack is the rate-limiting step. In the NADPH/oxygen-su pported cytochrome P450-catalyzed 4-hydroxylation of the anilines a co rrelation of the natural logarithm of k(cat)(S) with E(HOMO) was not o bserved and the k(cat)(S) values were lower than observed in the iodos obenzene-supported reaction. From this result it is concluded that, al though the NADPH/oxygen-supported microsomal 4-hydroxylation of the ha logenated anilines proceeds by the same cytochrome P450 (FeO)(3+) inte rmediate and, thus, by a similar electrophilic attack of the (FeO)(3+) on the pi electrons of the substrate, this attack is no longer the ra te-limiting step of the reaction. Additional results of the present st udy demonstrate that the apparent Michaelis constant K-m(S) of the NAD PH/oxygen-supported 4-hydroxylation of the anilines decreases with inc reasing hydrophobicity of the aniline derivatives. Because the spectra l dissociation constant K-d(S) Of the aniline-cytochrome P450 complex appeared to be severalfold lower than the K-m(S), it was concluded tha t other parameters than binding influence the K-m(S) of the cytochrome P450-catalyzed aniline 4-hydroxylation. In conclusion, the present pa per presents a MO-QSAR for the cytochrome P450-catalyzed conversion of a series of aniline derivatives. The implications of these findings f or the catalytic cycle of cytochrome P450 are being discussed.