Kinetic deuterium isotope effects in cytochrome P450 oxidation reactions

Abstract

Cytochrome P450 (P450) enzymes account for ~ 75% of the metabolism of drugs. Most of the reactions catalyzed by P450s are mixed-function oxidations, and a C-H bond is (usually) broken. The rate-limiting nature of this step can be analyzed using the kinetic isotope effect (KIE) approach. The most relevant type of KIE is one termed intermolecular non-competitive, indicative of rate-limiting C-H bond breaking. A KIE vs. kcat for several P450s showed a correlation coefficient (r2) of 0.62. Deuterium substitution has been considered as a potential means of slowing drug metabolism or redirecting sites of metabolism in some cases, and several general points can be made regarding the potential for application of deuterium in drug design/development based on what is known about P450 KIEs.

Mechanism of the P450-Catalyzed Oxidative Cyclization in the Biosynthesis of Griseofulvin

“…Guengerich states that it is generally accepted that for p450-catalyzed C-H abstraction mechanisms, if a KIE is observed then the C-H abstraction is partially rate limiting. 33 For this specific p450, the reduction of Fe 3+ to Fe 2+ during catalyst regeneration was found to be rate-limiting. 33,34 For many other p450 enzymes, the second reduction of the dioxygen-complex is rate limiting.…”

“…33 For this specific p450, the reduction of Fe 3+ to Fe 2+ during catalyst regeneration was found to be rate-limiting. 33,34 For many other p450 enzymes, the second reduction of the dioxygen-complex is rate limiting. 12,35,36 We suspect that a similar scenario is occurring with GsfF due to the low 3–5 kcal/mol barrier for the O-H abstractions and therefore another step in the enzymatically catalyzed reaction will be rate-limiting.…”

Aerobic biodegradation of 2 fluorotelomer sulfonamide–based aqueous film–forming foam components produces perfluoroalkyl carboxylates

“…The collision-induced dissociation spectra of products 2 and 3 in ESIþ are consistent with N-demethylations of 6:2 FTAA because they produce fragments of m/z 468 and 440 from loss of methylamine or ammonia followed by loss of ethene (Supplemental Data, Table S5 and Figure S7B,C), which are identical to the fragments produced by 6:2 FTAA [8]. Because this ion produces fragments with m/z 468 and 440 in ESIþ (Supplemental Data, Table S5 and Figure S7A), identical to 6:2 FTAA, corresponding to loss of a hydroxylated secondary amine and ethene, it can be inferred that the hydroxylation occurs on an a-methyl group as would be intermediate in either enzymatic or nonenzymatic N-demethylation [32,34,35]. Overall, N-demethylation appears to be favored in sterile controls over active experiments with larger LC-MS/ MS peaks for products 2 and 3 in sterile control extracts (Supplemental Data, Table S6).…”

The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1)

“…Deuterium-labeled substrates have been used for decades to study mechanistic and energetic aspects of enzymatic reactions involving C-H bond-breaking chemistry, such as cytochrome P450-catalyzed hydroxylations [23]. These experiments require access to the substrate bearing deuterium label at the bonds where chemistry occurs.…”