The Use of Deuterium Isotope Effects to Probe the Active Site Properties, Mechanism of Cytochrome P450-Catalyzed Reactions, and Mechanisms of Metabolically Dependent Toxicity

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Critical elements from studies that have led to our current understanding of the factors that cause the observed primary deuterium isotope effect, (k H /k D )obs, of most enzymatically mediated reactions to be much smaller than the "true" or intrinsic primary deuterium isotope effect, k H /k D , for the reaction are presented. This new understanding has provided a unique and powerful tool for probing the catalytic and active site properties of enzymes, particularly the cytochromes P450 (P450). Examples are presented that illustrate how the technique has been used to determine k H /k D , and properties such as the catalytic nature of the reactive oxenoid intermediate, prochiral selectivity, the chemical and enzymatic mechanisms of cytochrome P450-catalyzed reactions, and the relative active site size of different P450 isoforms. Examples are also presented of how deuterium isotope effects have been used to probe mechanisms of the formation of reactive metabolites that can cause toxic effects.Historically, the determination of deuterium isotope effects has been a powerful tool to help unravel the intricacies of carbon-hydrogen bond cleavage and define the mechanism of specific chemical reactions. The intrinsic primary isotope effect, k H /k D , for a reaction is the magnitude of the isotope effect on the rate constant for the bond-breaking step (C-H versus C-D) of the reaction and is related to the symmetry of the transition state for that step. The larger the isotope effect, the more symmetrical the transition state, with the theoretical limit being 9 at 37°C in the absence of tunneling effects (Bell, 1974). The chemical events leading to the transition state and subsequent formation of products are the descriptors of a chemical mechanism. It is the relationship of k H /k D to transition state that provides mechanistic insight. Thus, k H /k D is the quantity that needs to be known, and for homogenous chemical reactions, the experimentally observed deuterium isotope effect, (k H /k D )obs, where (k H /k D )obs is defined as the ratio of a kinetic parameter such as V max or V max /K m obtained from a nondeuterated substrate to a deuterated substrate, is identical to k H /k D . This is generally not true of enzymatically mediated reactions and is the reason for the much less successful application of deuterium isotope effects to such reactions until the system was better understood. The experimentally observed isotope effect, (k H /k D )obs, was invariably found to be much smaller than k H /k D , the intrinsic isotope effect for that reaction, thereby obscuring both meaning and mechanism. Even more perplexing was the observation that (k H /k D

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“…Another explanation may be found in the use of radiolabeled substrates, namely [20-2 H 3 ]taxa-4(5),11(12)-diene (17) or [20-3 H 3 ]taxa-4(5),11(12)-diene (8,16,17), in the initial characterization of CYP725A4 by Croteau and co-workers (16). Isotopic effects on P-450 enzymes are known to cause intramolecular competition leading to different products (35). Accordingly, the radioisotopes used may have prevented the normal sequence of electronic movements, leading to a diversion of the reaction.…”

Insights into the Catalytic Mechanisms of Phenylalanine and Tryptophan Hydroxylase from Kinetic Isotope Effects on Aromatic Hydroxylation

“…The theory of unmasking kinetic isotope effects by the partitioning of intermediates via branched pathways has been described previously (10,34,35). Scheme 3 illustrates the basic kinetic scheme for the analysis.…”

Differentiation of antiinflammatory and antitumorigenic properties of stabilized enantiomers of thalidomide analogs

“…The use of deuterium to stabilize drugs against undesirable metabolism, known as metabolic switching, began in the 1960s (46)(47)(48)(49)(50)(51) and is the predominant approach to deuterated drugs today. Metabolic switching can be a challenging strategy, because it is often difficult to translate from in vitro to in vivo (52), is limited to defined metabolic pathways, and requires the synthesis and testing of numerous analogs.…”