An assessment of the reaction energetics for cytochrome P450-mediated reactions

Regioselectivity is used to determine the absolute energetic differences fo r four different reactions catalyzed by P450, Abstraction of a hydrogen fro m a benzylic carbon containing a chlorine has a 1.0 kcal/mol lower barrier than abstraction from a simple benzylic carbon, which in turn is 0.4 to 0.9 kcal/mol lower than abstraction from the methyl group of an aromatic ether and 0.1 to 0.6 kcal/mol easier than aromatic hydroxylation. Isotope effect s are used to determine if the enzyme-substrate complexes leading to each p roduct, from a given substrate, are in rapid equilibrium, For all enzymes i sotopically sensitive branching is observed from the benzylic carbon upon d euterium incorporation at that position to each of the other positions, ind icating that each product arises from the same active oxygen species. The e nergetic differences determined experimentally are accurately reproduced by theoretical hydrogen atom abstractions at both the AM1 semiempirical and D FT levels of theory. (C) 2001 Academic Press.