Secondary Deuterium Kinetic Isotope Effects and Transition State Structure

Matsson, Olle; Westaway, Kenneth Charles

doi:10.1016/s0065-3160(08)60194-x

Cited by 45 publications

(65 citation statements)

References 165 publications

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“…The observed KIE for this enzyme fits the observed KIEs for other purine hydrolases where the 2 H-KIE has been observed to range from 1.08 to 1.17 (Berti and Tanake, 2002). The large effect is due to the increased freedom around C1 as it undergoes rehybridization in the formation of the oxocarbenium ion (Poirier et al, 1994;Koerner et al, 2000;Matsson and Westaway, 1998).…”

Section: Kinetic Isotope Effectssupporting

confidence: 82%

“…The large a secondary [1 0 -2 H]KIE has also been interpreted to be consistent with the formation of an oxocarbenium ion in which the C-N bond is significantly broken, but there is little bond order to the attacking nucleophile (Poirier et al, 1994;Koerner et al, 2000;Matsson and Westaway, 1998). The observed KIE for this enzyme fits the observed KIEs for other purine hydrolases where the 2 H-KIE has been observed to range from 1.08 to 1.17 (Berti and Tanake, 2002).…”

Section: Kinetic Isotope Effectssupporting

confidence: 75%

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Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus)

et al. 2006

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Section: Kinetic Isotope Effectssupporting

confidence: 82%

Section: Kinetic Isotope Effectssupporting

confidence: 75%

Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus)

et al. 2006

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“…The existence of a very high intrinsic kinetic deuterium isotope effect that is masked in other experimental settings is reminiscent of the work of Miwa et al (92) with some rat liver 4 We have not examined potential secondary isotope effects, which tend to be small (90). In principle, a careful comparison of reactions with 6,6-d 2 -and 6␤-d 1 -testosterone substrates might reveal a (geminal) secondary kinetic isotope effect.…”

Section: Regioselectivity Of Testosterone Oxidations Catalyzed By Biomentioning

confidence: 97%

Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps

Krauser

Guengerich

2005

Journal of Biological Chemistry

101

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Testosterone 6␤-hydroxylation is a prototypic reaction of cytochrome P450 (P450) 3A4, the major human P450. Biomimetic reactions produced a variety of testosterone oxidation products with 6␤-hydroxylation being only a minor reaction, indicating that P450 3A4 has considerable control over the course of steroid hydroxylation because 6␤-hydroxylation is not dominant in a thermodynamically controlled oxidation of the substrate. Several isotopically labeled testosterone substrates were prepared and used to probe the catalytic mechanism of P450 3A4: (i) 2,2,4,6,6-2 H 5 ; (ii) 6,6-2 H 2 ; (iii) 6␣-2 H; (iv) 6␤-2 H; and (v) 6␤-3 H testosterone. Only the 6␤-hydrogen was removed by P450 3A4 and not the 6␣, indicating that P450 3A4 abstracts hydrogen and rebounds oxygen only at the ␤ face. Analysis of the rates of hydroxylation of 6␤-1 H-, 6␤-2 H-, and 6␤-3 Hlabeled testosterone and application of the Northrop method yielded an apparent intrinsic kinetic deuterium isotope effect ( D k) of 15. The deuterium isotope effects on k cat and k cat /K m in non-competitive reactions were only 2-3. Some "switching" to other hydroxylations occurred because of 6␤-2 H substitution. The high D k value is consistent with an initial hydrogen atom abstraction reaction. Attenuation of the high D k in the non-competitive experiments implies that C-H bond breaking is not a dominant rate-limiting step. Considerable attenuation of a high D k value was also seen with a slower P450 3A4 reaction, the O-dealkylation of 7-benzyloxyquinoline. Thus P450 3A4 is an enzyme with regioselective flexibility but also considerable regioselectivity and stereoselectivity in product formation, not necessarily dominated by the ease of C-H bond breaking. P4501 enzymes are found in organisms from Archebacter to humans and catalyze oxidations of a great variety of chemicals (1, 2). The human P450s are the major enzymes involved in the clearance of drugs, and P450 3A4, the most abundant P450 in liver and small intestine, is involved in the oxidation of one-half of the drugs used today (3, 4). Two crystal structures of P450 3A4 have been published recently (5, 6); however, neither has a substrate bound in a position amenable to oxidation. P450 3A4 exhibits heterotropic and homotropic cooperativity in some but not all of its reactions (4, 7-10), and several models have been proposed to explain such behavior (9,(11)(12)(13)(14)(15). A view frequently expressed for smaller substrates is that binding is relatively loose and the regioselectivity of oxidation of substrates is largely a function of the ease of C-H bond breaking (16,17).Relatively little information is available regarding what step is rate-limiting in P450 3A4 reactions. The addition of the first electron in the catalytic cycle (step 2 of Scheme 1) is apparently relatively fast in the presence of an optimal concentration of NADPH-P450 reductase (18). Only limited information has been obtained regarding steps 1 and 3-9 (Scheme 1) with P450 3A4 reactions. Without information on the extent to which chemical step...

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“…Both reactions were highly dependent upon the presence of cytochrome b 5 ; with DMN (used at 17 mM), the rate of HCHO production was 0. (42). In the general literature, these are usually 1.0 -1.2 (42)(43)(44), including the few cases in which they have been estimated for P450s (45,46).…”

Section: Resultsmentioning

confidence: 99%

“…(42). In the general literature, these are usually 1.0 -1.2 (42)(43)(44), including the few cases in which they have been estimated for P450s (45,46). Such secondary isotope effects are multiplicative, so with two deuteriums, the contribution could be as high as 1.4.…”

Section: Resultsmentioning

confidence: 99%

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6

Chowdhury¹,

Calcutt²,

Guengerich

2010

Journal of Biological Chemistry

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Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH 3 CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect ( D k app ϳ 10), which was highly expressed in a variety of competitive and non-competitive experiments. The D k app for DEN was ϳ3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO 2 H and CH 3 CO 2 H, respectively. In neither case was a lag observed, which was unexpected considering the k cat and K m parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde). P4503 enzymes are found throughout nature and catalyze many reactions, most of which are mixed function oxidations (4). The mammalian P450s are of considerable interest because of their roles in the metabolism of steroids, eicosanoids, drugs, chemical carcinogens, and other important molecules (5). The general mechanistic features of P450 reactions include substrate binding, reduction to the ferrous state, binding of O 2 , the addition of a second electron, protonation, and rearrangement to generate a reactive iron-oxygen complex poised near the substrate (6, 7). The active complex can be described as a formal FeO 3ϩ entity, with similarity to Compound I of peroxidases, which can be used to rationalize most reactions (7-9), although some alternate possibilities can also be considered. A generally accepted mechanism for many P450 oxidations involves the abstraction of a hydrogen atom by the FeO 3ϩ entity, followed by "oxygen rebound" to yield a hydroxylated product (10).Among the chemical carcinogens activated by mammalian P450s are N,N-dialkylnitrosamines (also called N-nitrosodialkylamines) (11), including those found in tobacco products and also the simple nitrosamines N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN) (12). The mechanism of activation is agreed to involve ␣-hydroxylation of the nitrosamine in most cases (Fig. 1). The process is generally accepted to involve hydrogen atom transfer instead of the alternate 1e Ϫ oxidation implicated for some amines (7, 8,13,14) because ...

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Secondary Deuterium Kinetic Isotope Effects and Transition State Structure

Cited by 45 publications

References 165 publications

Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus)

Transition state analysis of adenosine nucleosidase from yellow lupin (Lupinus luteus)

Cytochrome P450 3A4-catalyzed Testosterone 6β-Hydroxylation Stereochemistry, Kinetic Deuterium Isotope Effects, and Rate-limiting Steps

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6

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