Abstract
Human class III alcohol dehydrogenase (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, exhibited non-hyperbolic kinetics with ethanol at a near physiological pH 7.5. The S 0.5 and k cat were determined to be 3.4±0.3 M and 33±3 min −1, and the Hill coefficient ( h) 2.21±0.09, indicating positive cooperativity. Strikingly, the S 0.5 for ethanol was found to be 5.4×10 6-fold higher than the K m for S-(hydroxymethyl)glutathione, a classic substrate for the enzyme, whereas the k cat for the former was 41% lower than that for the latter. Isotope effects on enzyme activity suggest that hydride transfer may be rate-limiting in the oxidation of ethanol. Kinetic simulations using the experimentally determined Hill constant suggest that gastric ADH3 may highly effectively contribute to the first-pass metabolism at 0.5–3 M ethanol, an attainable range in the gastric lumen during alcohol consumption. The positive cooperativity mainly accounts for this metabolic role of ADH3.
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