The separation of the intramolecular isotope effect for the cytochrome P-450 catalyzed hydroxylation of n-octane into its primary and secondary components

Jones, J. P.; Trager, William

doi:10.1021/ja00241a040

Cited by 44 publications

(36 citation statements)

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“…Thus, the enzyme always has the choice of oxidizing a carbonhydrogen or carbon-deuterium bond irrespective of which methyl group is oriented for catalysis. The value of (k H /k D )obs ϭ 16.11 for this substrate corresponds to an intrinsic primary isotope effect of 9.18 once it has been corrected for the contribution of secondary isotope effects and the fact that the methyl group contains two hydrogens but only one deuterium (Jones and Trager, 1987). As indicated above, an intrinsic primary isotope effect of 9 is the theoretical limit for cleavage of a carbon-hydrogen bond in the absence of tunneling effects (Bell, 1974).…”

Section: Symmetrical Intramolecular Designmentioning

confidence: 99%

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

Nelson¹,

Trager²

2003

Drug Metab Dispos

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141

121

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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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Section: Symmetrical Intramolecular Designmentioning

confidence: 99%

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

Nelson¹,

Trager²

2003

Drug Metab Dispos

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141

121

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“…Geminal secondary isotope effects are much lower than primary isotope effects (Յ1.4) (73,86). Relatively few secondary deuterium isotope effects have been measured in P450 reactions (87)(88)(89), and some of these were measured with crude microsomal systems, not individual enzymes (87,88 …”

mentioning

confidence: 99%

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Yun

Kim

Calcutt

et al. 2005

Journal of Biological Chemistry

115

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Human cytochrome P450 (P450) 2A6 catalyzes 7-hydroxylation of coumarin, and the reaction rate is enhanced by cytochrome b 5 (b 5 ). 7-Alkoxycoumarins were O-dealkylated and also hydroxylated at the 3-position. Binding of coumarin and 7-hydroxycoumarin to ferric and ferrous P450 2A6 are fast reactions (k on ϳ 10 6 M ؊1 s ؊1 ), and the k off rates range from 5.7 to 36 s ؊1 (at 23°C). Reduction of ferric P450 2A6 is rapid (7.5 s ؊1 ) but only in the presence of coumarin. The reaction of the ferrous P450 2A6 substrate complex with O 2 is rapid (k > 10 6 M ؊1 s ؊1 ), and the putative Fe 2؉ ⅐O 2 complex decayed at a rate of ϳ0.3 s ؊1 at 23°C. Some 7-hydroxycoumarin was formed during the oxidation of the ferrous enzyme under these conditions, and the yield was enhanced by b 5 . Kinetic analyses showed that ϳ 1 ⁄3 of the reduced b 5 was rapidly oxidized in the presence of the Fe 2؉ ⅐O 2 complex, implying some electron transfer. High intrinsic and competitive and non-competitive intermolecular kinetic deuterium isotope effects (values 6 -10) were measured for O-dealkylation of 7-alkoxycoumarins, indicating the effect of C-H bond strength on rates of product formation. These results support a scheme with many rapid reaction steps, including electron transfers, substrate binding and release at multiple stages, and rapid product release even though the substrate is tightly bound in a small active site. The inherent difficulty of chemistry of substrate oxidation and the lack of proclivity toward a linear pathway leading to product formation explain the inefficiency of the enzyme relative to highly efficient bacterial P450s. P4501 enzymes are involved in the oxygenation of a variety of natural products and xenobiotic chemicals in microbial systems (3, 4). Much is known about the structure, function, and catalytic features of some of the P450s, particularly the more extensively studied of the bacterial P450s (4, 5). In mammalian systems P450s oxidize many drugs, steroids, carcinogens, fatty acids and eicosanoids, fat-soluble vitamins, and other endobiotic and xenobiotic chemicals (6). Less information is available about the biochemical details of most of the 57 human P450s (7). In particular, the basis of the inherently lower catalytic activities of these and other mammalian P450s relative to some of the microbial forms is not clear.P450 2A6 is a low-to-medium abundance P450 in human liver (7-9) and is also expressed in some extrahepatic tissues (10). The history of this gene/protein goes back to Phillips et al. (11), who identified a human P450 cDNA as a relative of rat P450 2B1. The 7-hydroxylation of coumarin has long been used as an assay of P450 activity in animal and human liver microsomes (12, 13), and Yamano et al. (14) isolated a P450 2A6 cDNA (then termed 2A3) and first showed that the protein derived from heterologous expression had coumarin 7-hydroxylation activity. Miles et al. (15) also provided similar evidence for this particular sequence being associated with coumarin 7-hydroxylation. Our group purified a pr...

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“…It has been established that oxidation of the terminal methyl group of octane has a reasonably symmetrical reaction coordinate based on observed primary and secondary isotope effects. 15 N-dealkylation reactions are relatively more exothermic and show smaller isotope effects than aliphatic oxidation reactions. 16 Thus, Cpd 0, which has been established to be less reactive than Cpd I, 6,8,11 would react more endothermically and produce higher isotope effects.…”

mentioning

confidence: 99%

Kinetic Isotope Effects Implicate the Iron−Oxene as the Sole Oxidant in P450-Catalyzed N-Dealkylation

Dowers

Rock

et al. 2004

J. Am. Chem. Soc.

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Multiple oxidants have been implicated as playing a role in cytochrome P450-mediated oxidations. Herein, we report results on N-dealkylation, one of the most facile reactions mediated by P450 enzymes. We have employed the N-oxides of a series of para-substituted 13C2H2-labeled N,N-dimethylanilines to function as both substrates and surrogate oxygen atom donors for P450cam and P4502E1. Kinetic isotope effect profiles obtained using the N-oxide system were found to closely match the profiles produced using the complete NAD(P)H/NAD(P)-P450 reductase/O2 system. The results are consistent with oxidation occurring solely through an iron-oxene species.

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The separation of the intramolecular isotope effect for the cytochrome P-450 catalyzed hydroxylation of n-octane into its primary and secondary components

Cited by 44 publications

References 2 publications

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

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

Kinetic Analysis of Oxidation of Coumarins by Human Cytochrome P450 2A6

Kinetic Isotope Effects Implicate the Iron−Oxene as the Sole Oxidant in P450-Catalyzed N-Dealkylation

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