Evaluation of Cytochrome P450 Mechanism and Kinetics Using Kinetic Deuterium Isotope Effects

Higgins, LeeAnn; Bennett, G. A.; Shimoji, Miyuki; Jones, Jeffrey P.

doi:10.1021/bi9729864

Cited by 30 publications

(26 citation statements)

References 36 publications

(52 reference statements)

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“…We have seen similar results for the C-1 hydroxylation of octane; however in this case, the alternate products were C-2 and C-3 oxidation products (Jones et al, 1986). Therefore, slowing the rate of oxidation of one position by deuterium substitution causes an increase in the rate of metabolism of another position or an increase in decoupling to water formation (Higgins et al, 1998). This can only occur if the rate of exchange of the different positions within the active site is as fast or faster than the rate of the oxidation step.…”

Section: Discussionsupporting

confidence: 64%

“…In addition, results show that useful correlations between the rates of product formation can be developed using electronic models (Grogan et al, 1992;Yin et al, 1995). These results are apparently contradicted by the results of kinetic experiments, which show the steps before substrate oxidation (electron reduction) to be rate limiting in the catalytic cycle (Higgins et al, 1998). This apparent contradiction can be resolved by an understanding of the kinetics of branched pathways, which was described by Jones et al (1986).…”

Section: Discussionmentioning

confidence: 99%

“…The combined models allow for the prediction of the electronic component for the regioselectivity of most of the reactions catalyzed by the cytochrome P450 superfamily of enzymes. These models are general models for the electronic component of P450 enzymes, which we have shown to be rather constant across both the enzyme families and species (Jones et al, 1990;Karki et al, 1995;Yin et al, 1995;Higgins et al, 1998).…”

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confidence: 99%

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Computational Models for Cytochrome P450: A Predictive Electronic Model for Aromatic Oxidation and Hydrogen Atom Abstraction

Jones¹,

Mysinger²,

Korzekwa³

2002

Drug Metabolism and Disposition

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This paper is available online at http://dmd.aspetjournals.org ABSTRACT:Experimental observations suggest that electronic characteristics play a role in the rates of substrate oxidation for cytochrome P450 enzymes. For example, the tendency for oxidation of a certain functional group generally follows the relative stability of the radicals that are formed (e.g., N-dealkylation > O-dealkylation > 2°c arbon oxidation > 1°carbon oxidation). In addition, results show that useful correlations between the rates of product formation can be developed using electronic models. In this article, we attempt to determine whether a combined computational model for aromatic and aliphatic hydroxylation can be developed. Toward this goal, we used a combination of experimental data and semiempirical molecular orbital calculations to predicted activation energies for aromatic and aliphatic hydroxylation. The resulting model extends the predictive capacity of our previous aliphatic hydroxylation model to include the second most important group of oxidations, aromatic hydroxylation. The combined model can account for about 83% of the variance in the data for the 20 compounds in the training set and has an error of about 0.7 kcal/mol. The P4501 enzymes are a superfamily of monooxygenases involved in the metabolism of both exogenous and endogenous compounds. Ironically, these enzymes play a central role in both the prevention and induction of chemical toxicities and carcinogenicity. Although most P450 oxidations of xenobiotics result in detoxification, occasionally a more toxic intermediate is formed. In fact, many ultimate toxins and carcinogens are formed by the bioactivation of less reactive compounds, and many bioactivation reactions are mediated by the P450 enzymes. Often bioactivation reactions are in competition with detoxification pathways for the same substrate. Since these enzymes play such a central role in both detoxification and bioactivation, predictive models for cytochrome P450 catalysis will be useful tools for evaluating of the potential risks of environmental exposures. One of the most pertinent but difficult problems in risk assessment is translating bench results and mechanistic information into a form that can be used. This article outlines steps toward the development of computational models from laboratory data into a form that can be used for risk assessments that accurately reflect experimental results. For example, these models now more completely describe all positions of metabolism for nitriles and should be more complete in predicting the toxicity related to nitrile metabolism. These semiempirical computational models blend experimental data and computational chemistry in such a way as to provide a consistent prediction of the bioactivation rates for a broad spectrum of compounds. In particular, these models can be used to predict xenobiotic metabolism by the P450 enzyme family, including the bioactivation of compounds to toxins and carcinogens.Models such as those presented here for P450-mediated reaction...

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Section: Discussionsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Computational Models for Cytochrome P450: A Predictive Electronic Model for Aromatic Oxidation and Hydrogen Atom Abstraction

Jones¹,

Mysinger²,

Korzekwa³

2002

Drug Metabolism and Disposition

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120

132

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“…Such a branch point leads to the "unmasking" of the isotope effect. Partial reduction of the activated perferryl species to water can have the same effect Higgins et al, 1998). Thus, water formation as well as minor switching to TB and TP facilitated the observation of isotope effects on D V and D V/K.…”

Section: Discussionmentioning

confidence: 95%

“…Formation of multiple metabolites, as a result of rapid equilibration of the multiple EOS complexes, allows the observation of isotope effects within D V/K by providing a siphon for the excess EOS D (Jones et al, 1986;Korzekwa et al, 1989;Higgins et al, 1998;Nelson and Trager, 2003). Such a branch point leads to the "unmasking" of the isotope effect.…”

Section: Discussionmentioning

confidence: 99%

Oxidation of Caffeine by Cyp1a2: Isotope Effects and Metabolic Switching

Regal

Kunze

Peter

et al. 2005

Drug Metabolism and Disposition

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ABSTRACT:Caffeine (1,3,7-trimethylxanthine) has previously been shown to undergo metabolic switching in vivo when the N-1 or the N-7 methyl groups were trideuteromethylated [Horning et al. (1976) Proceedings of the Second International Conference on Stable Isotopes, pp 41-54]. We have examined the effect of replacing the N-3 methyl group with a trideuteromethyl group. The corresponding isotope effects can then be used to distinguish the kinetic mechanism by which four primary metabolites can be formed from one substrate by one cytochrome P450 (P450). We have synthesized 3-CD 3 -caffeine and 3-CD 3 -7-CD 3 -caffeine as well as trideuteromethylated analogs of each of the in vitro metabolites formed by cytochrome P4501A2. The observed competitive isotope effects for the metabolites, which do not result from deuterium abstraction (theobromine, theophylline), demonstrate that the nondissociative mechanism applies to caffeine metabolism by cytochrome P4501A2. Thus, there must be equilibration of the kinetically distinguishable activated P450-substrate complexes at rates competitive with hydrogen abstraction. The true isotope effects for the N-3 demethylation of caffeine were derived from the ratios of the amount of paraxanthine relative to the amount of theobromine or theophylline. The resultant ratios indicate that these isotope effects are essentially intrinsic. Observation of the isotope effects on N-3 demethylation was facilitated by branching to the minor in vitro metabolites as well as water formation. Product release is not rate-limiting for this system. Cytochrome P4501A2 (CYP1A2) is estimated to be responsible for 90% of the primary metabolism of caffeine in humans (Tassaneeyakul et al., 1994) and is believed to be largely responsible for formation of the major metabolite, paraxanthine (PX), in vivo. The four in vitro metabolites formed by human CYP1A2 are PX (80%), theobromine (TB; 11%), theophylline (TP; 4%) and 1,3,7-trimethyluric acid (TMU; 1%) ( Fig. 1; Gu et al., 1992). Presumably, multiple binding orientations exist within the active site of CYP1A2 since the entire periphery of caffeine is available for metabolism.Experimental evidence suggests that when metabolically susceptible hydrogen(s) are replaced with deuterium atoms, there can be a decrease in the oxidation rate at the labeled site with no concomitant change in the overall extent of metabolism (Harada et al., 1984;Atkins and Sligar, 1986;Jones et al., 1986). Thus, the net effect of deuteration is an increased rate of formation of one or all of the alternate metabolites, a phenomenon known as "isotopically sensitive branching" or "metabolic switching." On the basis of studies that exhibited classical metabolic switching (Harada et al., 1984), it was hypothesized that switching occurs at the level of the activated P450 or "EOS complex," and not earlier in the catalytic cycle. Theoretical studies have supported this concept (Korzekwa et al., 1989;Nelson and Trager, 2003). Hence, deuterium-induced metabolic switching indicates that isomeric EOS co...

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Formation of the Active Species of Cytochrome P450 by Using Iodosylbenzene: A Case for Spin‐Selective Reactivity

Cho

Moreau

Kumar

et al. 2007

Chemistry A European J

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Part 4: Theoretical KIE calculations Part 5: Experimental section S3 MethodsOptimizations have been done on B3LYP/LACVP level, and single point LACV3P*+ on the optimized geometry where indicated (ERMLER2*+ on iodine). Frequency and single point solvent calculations were also done on the optimized geometry using LACVP. Spin and charge values are taken from big basis set calculations. values are from LACVP calculations. Remarks about the accuracy of B3LYPAs can be seen from the extensive data in the text and elsewhere in the literature, UB3LYP predicts the correct ground state for the systems of this study. Here are a few additional comments:• S5 Part 1 Reaction between Heme and IodosylbenzeneIn this summary, the five different stages of the reaction are named as below:Heme + OIPh: Heme and Iodosylbenzene, uncomplexed.

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Evaluation of Cytochrome P450 Mechanism and Kinetics Using Kinetic Deuterium Isotope Effects

Cited by 30 publications

References 36 publications

Computational Models for Cytochrome P450: A Predictive Electronic Model for Aromatic Oxidation and Hydrogen Atom Abstraction

Computational Models for Cytochrome P450: A Predictive Electronic Model for Aromatic Oxidation and Hydrogen Atom Abstraction

Oxidation of Caffeine by Cyp1a2: Isotope Effects and Metabolic Switching

Formation of the Active Species of Cytochrome P450 by Using Iodosylbenzene: A Case for Spin‐Selective Reactivity

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