Experimental and theoretical study of the effect of active-site constrained substrate motion on the magnitude of the observed intramolecular isotope effect for the P450 101 catalyzed benzylic hydroxylation of isomeric xylenes and 4,4′-dimethylbiphenyl

The validity of a cytochrome P450 (P450) 101 force field developed previously was tested by comparing to published results from other laboratories the predicted regioselectivity and stereoselectivity of both (R)- and (S)-norcamphor oxidation when the force field was used. Once validated, the force field was used to test the hypothesis that the magnitude of an observed intramolecular isotope effect is a function of the distance between equivalent but isotopically distinct intramolecular sites of oxidative attack. Molecular dynamics simulations and kinetic deuterium isotope effect experiments on benzylic hydroxylation were then conducted for a series of selectively deuterated isomeric xylenes and 4,4'-dimethylbiphenyl with P450 101. The molecular dynamics simulations predicted that the rank order of substrate mobility in the active site of P450 101 was o-xylene > p-xylene > dimethylbiphenyl. The observed isotope effects for the trideutero analogues were 10.6, 7.4, and 2.7, for the o-xylene, p-xylene, and 4,4'-dimethylbiphenyl, respectively. Thus, as the theoretically predicted rates of interchange between the isotopically distinct methyl groups decrease, the observed isotope effect decreases. The agreement between the theoretical predictions and experimental results provides strong support for the distance hypothesis stated above and for the potential of computational analysis to enhance our understanding of protein/small molecule interactions.