Received Sep 8; Accepted Oct
RH, substrate, ROH, product. The reversibility of some of the latter steps is unknown. As pointed out earlier 10with P 2E1 the binding of substrate can followstep 2 and possibly 3 and4. P 2E1 is considered to be one of the major human hepatic P enzymes Human P 2E1, as well as the animal orthologs, accepts a broad range of substrates, with apparent preference for small and hydrophobic molecules 414 P 2E1 is notably active in the oxidation of many low M r volatile solvents with common industrial applications and issues of cancer risk 16 Many P 2E1 reactions have been characterized, at least in terms of the products 1718and the Michaelis-Menten constants have been determined.
Some reactions have been further studied by employing isotopic substitution of the substrate.
One example is the P 2E1-catalyzed oxidation of ethanol to acetaldehyde 10 This laboratory, as well as others, has indicated that this kinetic hydrogen isotope effect can been explained by rate-limiting product release following the isotopically sensitive and essentially irreversible C—H bond-breaking step 1920and experimental evidence has been offered in support of this model A carbonyl product is generated in each of the oxidation reactions displaying this pattern of isotope effects 19 We have used steady-state, rapid quench, and pulse-chase kinetic experiments to analyze the sequential oxidations that convert ethanol to acetaldehyde and, ultimately, to acetic acid.
Recombinant human P 2E1 was expressed in Escherichia coli and purified essentially as described Chemicals Acetaldehyde, acetic acid, and 4-methylpyrazole were purchased from Aldrich; acetaldehyde was purified by distillation at atmospheric pressure prior to use.
Stock solutions of 50 mm 4-methylpyrazole in H2O were used in binding studies. Reagent grade ethanol was obtained from McCormick Distilling Co. Baker, Phillipsburg, NJ to remove components that might interfere with [14C]acetic acid and [14C]acetaldehyde determinations Synthesis of [H,C]Acetaldehyde [H,C]Acetaldehyde was synthesized using pyruvate decarboxylase, an enzyme that converts pyruvic acid to acetaldehyde with the addition of a solvent proton retained at the aldehyde C-1 position All reagents were dissolved in 2H2O and purged with argon.
The reaction solution contained 10 units of pyruvate decarboxylase dialyzed versus 2H2O as described above1. The disappearance of [14C]pyruvic acid was monitored by ion pair HPLC essentially as described 32except that an isocratic mobile phase of 10 mmtetra-n-butylammonium hydrogen sulfate, pH 6.
The major radioactive fractions were pooled, and the [H,C]acetaldehyde concentration was determined by liquid scintillation spectrometry based on the 14C specific activity of The identity of the product was confirmed by derivatization with 2,4-dinitrophenylhydrazine 33 followed by NMR and electrospray mass spectrometry.
Aldehyde Oxidation Assays P 2E1 1. After loading, the reaction mixtures were applied, the columns were washed with 3 column volumes of H2O, and the [14C]acetic acid product was eluted with 2.
The radioactive product was collected and counted by liquid scintillation spectrometry, with calibration of counting efficiency using external 14C-toluene standards.
Ethanol 20 mm was added to the reaction mixture. Acetic acid 0—50 mm, as indicated was included as the competitive ligand.
The acetaldehyde product was derivatized to form the 2,4-dinitrophenylhydrazone 33 and then analyzed by HPLC using a Zorbax 6. Acetic acid generated from [C]ethanol was quantitated as described above.
Apparent Isotope Effect Determination Deuterium isotope effects were determined by a noncompetitive method 2. P 2E1 was incubated with unlabeled d 0 acetaldehyde or [H]acetaldehyde d 1and the products were analyzed as described. In a similar reaction, ethanol oxidation was initiated by the addition of ethanol to reconstituted P 2E1 The latter reaction is again catalyzed by an alcohol dehydrogenase, now operating in the opposite direction.
Uses Acetaldehyde, derived from mucosal or microbial oxidation of ethanol, tobacco smoke, and diet, appears to act as a cumulative carcinogen in the upper digestive tract of humans.
S1 Supporting Information for Mechanism of Copper/Azodicarboxylate-Catalyzed Aerobic Alcohol Oxidation: Evidence for Uncooperative Catalysis. The present paper summarizes density functional theory studies on hydroxylation of cyclohexene catalyzed by manganese oxo porphyrin.
The reaction is preceded by the physisorption of the substrate over the catalyst molecule at the distance of Å. Lignin valorization has long been recognized as a sustainable solution for the renewable production of aromatic compounds. Two-step oxidation/reduction strategies, whereby the first oxidation step is required to “activate” lignin systems for controlled fragmentation reactions, have recently emerged as .
Communication Dual Catalysis: Combination of Photocatalytic Aerobic Oxidation and Metal Catalyzed Alkynylation Reactions—C C Bond Formation Using Visible Light. the Thermal-Catalytic Oxidation of Ethanol by TiO 2 during the photocatalytic degradation of selected organics, while Scott et al.
found that preilluminating Pt/TiO 2 with UV light improved the catalytic oxidation of formic acid by a factor of ,6 In a separate study.