ACTIVE-SITE DYNAMICS OF TOLUENE HYDROXYLATION BY CYTOCHROME-P-450

Rat liver cytochrome P-450 hydroxylates toluene to benzyl alcohol plus o-, m-, and p-cresol. Deuterated toluenes (C6D5CH3, C6D5CD3, PhCDnH3- n) were incubated under saturating conditions with liver microsomes from phenobarbital-pretreated rats, and product yields and ratios were measured. Stepwise deuteration of the methyl leads to stepwise decreases in the alcohol/cresol ratio without changing the cresol isomer ratios. Extensive deuterium retention in the benzyl alcohols from PhCH2D and PhCHD2 suggests there is a large intrinsic isotope effect for benzylic hydroxylation. After replacement of the third benzylic H by D, the drop in the alcohol/cresol ratio was particularly acute, suggesting that metabolic switching from D to H within the methyl group was easier than switching from the methyl to the ring. Comparison of the alcohol/cresol ratio for PhCH3 vs PhCD3 indicated a net isotope effect of 6.9 for benzylic hydroxylation. From product yield data for PhCH3 and PhCD3, DV for benzyl alcohol formation is only 1.92, whereas DV for total product formation is 0.67 (i.e., inverse). These observations can be explained by postulating that product release is partly rate limiting in turnover when the product is benzyl alcohol but not when it is cresol; thus, deuterium-induced metabolic switching directs the substrate to a pathway (ring hydroxylation) that is intriniscally harder to enter but ultimately faster to traverse. Quantitative fitting of the data to a kinetic model of this situation supports these assumptions and indicates a net intrinsic kinetic isotope effect (£hh/&dd)of or benzylic hydroxylation of toluene. From competitive incubations of PhCH3/PhCD3 mixtures D(V/K) isotope effects on benzyl alcohol formation and total product formation (3.6 and 1.23, respectively) are greatly reduced, implying strong commitment to catalysis. In contrast, D(V/K) for the alcohol/cresol ratio is 6.3, indicating that the majority of the intrinsic isotope effect is expressed through metabolic switching. Overall, these data are consistent with reversible formation of a complex between toluene and the active oxygen form of cytochrome P-450, which rearranges internally and reacts to form products faster than it dissociates back to release substrate; the benzylic position is more reactive than the aromatic ring, but release of benzyl alcohol is much slower than release of cresol (or its arene oxide precursor) and is partially rate limiting in turnover. © 1990, American Chemical Society. All rights reserved.