Specificity of human alcohol dehydrogenase 1C*2 (gamma2gamma2) for steroids and simulation of the uncompetitive inhibition of ethanol metabolism.
The steady-state kinetics of the recombinant human alcohol dehydrogenase (ADH) 1C*2 with steroids were studied in order to determine substrate and inhibitor specificity. The assays were carried out under conditions of pH and temperature that are similar to those found in vivo. The enzyme has measurable activity on 5beta-androstan-17beta-ol-3-one, 5beta-androstan-3beta-ol-17-one, 5beta-pregnan-3beta-ol-20-one and 5beta-pregnan-3,20-dione, but much less activity with 5beta-cholanic acid-3-one or 5alpha-pregnan-3beta-ol-20-one. The determinants of specificity appear to include a 5beta configuration (cis A/B ring fusion) and a 3beta-hydroxy or 3-keto group. None of the reactive steroids has a known function in vivo. The activities with the human ADH1C*2 are <10% of those found with the recombinant horse ADH1S, but higher than the activities with recombinant horse ADH1E, which has an active site very similar to human ADH1C. 5alpha-Dihydrotestosterone is a ketone and a competitive inhibitor against varied concentrations of the substrate cyclohexanone, whereas it is an uncompetitive inhibitor against ethanol or NAD(+). Such patterns are expected for the binding of the steroid as a dead-end inhibitor to the enzyme-NADH complex. Thus, it does not appear that 5alpha-dihydrotestosterone is an allosteric inhibitor of the enzyme. Another dead-end inhibitor that gives uncompetitive inhibition of alcohol oxidation, 3-butylthiolane 1-oxide, is a potent inhibitor of alcohol metabolism in rats and mice. Simulation of the kinetics of ethanol elimination in rats with varied concentrations of the inhibitor is shown to yield the in vivo inhibition constant and an estimate of the rate of elimination of the inhibitor.
Plapp
Bryce V
BV
Department of Biochemistry, The University of Iowa, Iowa City, IA 52242-1109, USA. bv.plapp@uiowa.edu
Berst
Kristine B
KB
D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013256:Steroids; D000431:Ethanol; D000426:Alcohol Dehydrogenase