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 previous ly was tested by comparing to published results from other laboratories the predicted regioselectivity and stereoselectivity of both (R)- and (S)-norc amphor oxidation when the force field was used. Once validated, the force f ield was used to test the hypothesis that the magnitude of an observed intr amolecular isotope effect is a function of the distance between equivalent but isotopically distinct intramolecular sites of oxidative attack. Molecul ar dynamics simulations and kinetic deuterium isotope effect experiments on benzylic hydroxylation were then conducted for a series of selectively deu terated isomeric xylenes and 4,4'-dimethylbiphenyl with P450 101. The molec ular dynamics simulations predicted that the rank order of substrate mobili ty in the active site of P450 101 was o-xylene > p-xylene > dimethylbipheny l. 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, respectiv ely. Thus, as the theoretically predicted rates of interchange between the isotopically distinct methyl groups decrease, the observed isotope effect d ecreases. The agreement between the theoretical predictions and experimenta l results provides strong support for the distance hypothesis stated above and for the potential of computational analysis to enhance our understandin g of protein/small molecule interactions.