Kinetics of Cytochrome P450 2E1-Catalyzed Oxidation of Ethanol to Acetic Acid via Acetaldehyde

Bell-Parikh, L. Chastine; Guengerich, F. Peter

doi:10.1074/jbc.274.34.23833

Cited by 142 publications

(138 citation statements)

References 51 publications

(55 reference statements)

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“…When reconstituted with NADPH-cytochrome P-450 reductase and cytochrome b 5 , the truncated, purified CYP2E1 is catalytically competent in performing two classic CYP2E1 reactions: 2-hydroxylation of p-nitrophenol and 6-hydroxylation of chlorzoxazone (data not shown). Additionally, this enzyme has K d values for INZ and 4MP similar to those previously reported for the full-length rabbit CYP2E1 (41,42). Diffraction data were collected to 2.2 Å on a single crystal of CYP2E1 co-crystallized with INZ and to 2.6 Å on a single crystal of CYP2E1 co-crystallized with 4MP.…”

Section: Resultsmentioning

confidence: 99%

Structures of Human Cytochrome P-450 2E1

Porubsky¹,

Meneely²,

Scott

2008

Journal of Biological Chemistry

195

102

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Human microsomal cytochrome P-450 2E1 (CYP2E1) monooxygenates >70 low molecular weight xenobiotic compounds, as well as much larger endogenous fatty acid signaling molecules such as arachidonic acid. In the process, CYP2E1 can generate toxic or carcinogenic compounds, as occurs with acetaminophen overdose, nitrosamines in cigarette smoke, and reactive oxygen species from uncoupled catalysis. Thus, the diverse roles that CYP2E1 has in normal physiology, toxicity, and drug metabolism are related to its ability to metabolize diverse classes of ligands, but the structural basis for this was previously unknown. Structures of human CYP2E1 have been solved to 2.2 Å for an indazole complex and 2.6 Å for a 4-methylpyrazole complex. Both inhibitors bind to the heme iron and hydrogen bond to Thr 303 within the active site. Complementing its small molecular weight substrates, the hydrophobic CYP2E1 active site is the smallest yet observed for a human cytochrome P-450. The CYP2E1 active site also has two adjacent voids: one enclosed above the I helix and the other forming a channel to the protein surface. Minor repositioning of the Phe 478 aromatic ring that separates the active site and access channel would allow the carboxylate of fatty acid substrates to interact with conserved 216 QXXNN 220 residues in the access channel while positioning the hydrocarbon terminus in the active site, consistent with experimentally observed -1 hydroxylation of saturated fatty acids. Thus, these structures provide insights into the ability of CYP2E1 to effectively bind and metabolize both small molecule substrates and fatty acids.Cytochrome P-450 (P-450) 3 is a superfamily of enzymes involved in monooxygenation of both endogenous and exogenous substrates. A subset of these enzymes, including cytochrome P-450 2E1 (CYP2E1), are known for their role in the clearance of drugs and other xenobiotics by introducing or unmasking sites for subsequent conjugation and elimination from the body. From a biochemical standpoint these enzymes are fascinating for the diversity of substrates each xenobioticmetabolizing P-450 can metabolize, yet often with exquisite selectivity in the metabolites generated. Unfortunately, in the process some P-450-mediated metabolism can produce toxic or carcinogenic products. Among cytochrome P-450 enzymes, CYP2E1 is particularly notable for this ability and the resulting toxicity (1). This activity is most substantial in the liver because CYP2E1 comprises over 50% of the hepatic cytochrome P-450 mRNA (2) and 7% of the hepatic cytochrome P-450 protein (3). However, CYP2E1 is also expressed at lower levels in a variety of extrahepatic tissues (4), where it is thought to play a role in the metabolism of important endogenous molecules. CYP2E1 levels and the resulting toxicity varies markedly in response to alcohol consumption (5), diabetes (6), obesity (7), and fasting (8). Thus, the action of CYP2E1 can have a significant influence on human health and drug metabolism.CYP2E1 has been connected with liver toxicity through two ...

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Section: Resultsmentioning

confidence: 99%

Structures of Human Cytochrome P-450 2E1

Porubsky¹,

Meneely²,

Scott

2008

Journal of Biological Chemistry

195

102

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“…The C-H bond-breaking step appears to be less rate-limiting in P450 3A4 reactions than with several reactions catalyzed by other mammalian P450s, e.g. P450s 1A2 (42,44), 2A6 (45), 2E1 (41,103), and 2D6 (43). These results, coupled with the very selective stereochemistry of hydrogen abstraction (Fig.…”

Section: Regioselectivity Of Testosterone Oxidations Catalyzed By Biomentioning

confidence: 99%

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

Krauser

Guengerich

2005

Journal of Biological Chemistry

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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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“…Indoline-2,3-diol may be unstable (51), but it could be oxidized by H 2 O 2 and IDO-Fe 3+ to form the stable products II and/or IV. The mechanism of this subsequent oxidation is speculative but may involve P450-type ferryl chemistry relevant to C-C bond cleavage of diols (26,52) and alcohol oxidation (53,54) (Fig. S4).…”

Section: •−mentioning

confidence: 99%

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Kuo

Mauk

2012

Proc. Natl. Acad. Sci. U.S.A.

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The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to catalyze the oxidation of indole by H 2 O 2 , with generation of 2-and 3-oxoindole as the major products. This reaction occurred in the absence of O 2 and reducing agents and was not inhibited by superoxide dismutase or hydroxyl radical scavengers, although it was strongly inhibited by L-Trp. The stoichiometry of the reaction indicated a one-to-one correspondence for the consumption of indole and H 2 O 2 . The 18 O-labeling experiments indicated that the oxygen incorporated into the monooxygenated products was derived almost exclusively from H 2 18 O 2 , suggesting that electron transfer was coupled to the transfer of oxygen from a ferryl intermediate of IDO. These results demonstrate that IDO oxidizes indole by means of a previously unrecognized peroxygenase activity. We conclude that IDO inserts oxygen into indole in a reaction that is mechanistically analogous to the "peroxide shunt" pathway of cytochrome P450.monooxygenase | oxygen isotopic labeling T he heme enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-determining step of L-tryptophan metabolism in nonhepatic mammalian tissues by inserting both atoms of O 2 into the indole ring to form N-formylkynurenine (N-FK) (1). This dioxygenase activity of IDO requires O 2 and the reduced (Fe 2+ ) enzyme or O 2•− and the oxidized (Fe 3+ ) enzyme. In recent years, an increasing number of physiological consequences of IDO activity have been identified, including links to neural, ocular, and immunological disorders (2). Of these roles, the ability of IDO expressed by tumors to suppress the normal response of T lymphocytes and enable tumor survival and growth has attracted the most intense interest, owing to its implications for the development of new therapeutic approaches in cancer treatment (3).Aside from tryptophan (Trp), the dioxygenase activity of IDO extends to oxidation of other indoleamines, such as 5-hydroxy-LTrp, serotonin, and tryptamine, whereas indole, 3-methylindole, and indoleacetic acid are not substrates (4-6). Although indole can apparently bind to the oxygenated enzyme (IDOFe 3+ -O 2•− ) or to the IDOFe 2+ -CO complex (7), autoxidation to IDOFe 3+ is unaffected by the presence of indole, and oxidation of indole does not occur (5). Notably, IDO does not catalyze oxidation of L-Trp by H 2 O 2 (4, 8, 9). Consequently, the reactivity of IDO with H 2 O 2 received little attention for more than 30 years. During that time, IDO was noted to have peroxidase activity (6) and to catalyze the H 2 O 2 -supported N-demethylation of benzphetamine and hydroxylation of aniline (10), but these activities were not studied further.Recently, reactivity of the enzyme with H 2 O 2 has received considerable attention in light of spectroscopic detection of a compound II-like ferryl species (11, 12) and quantum mechanics/ molecular mechanics simulations implicating a role for this intermediate in the catalytic cycle (13). Subsequently, the peroxidase activity of IDO has received renewed atten...

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Kinetics of Cytochrome P450 2E1-Catalyzed Oxidation of Ethanol to Acetic Acid via Acetaldehyde

Cited by 142 publications

References 51 publications

Structures of Human Cytochrome P-450 2E1

Structures of Human Cytochrome P-450 2E1

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

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

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