| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 89 === Alcohol dehydrogenase (ADH), is the principal enzyme responsible for metabolism of ethanol in human body. The purpose of this thesis was to (a) determine kinetic parameters for ethanol oxidation for the recombinant human ADH family members; (b) explore kinetic interactions between ethanol and aspirin; and (c) simulate in vivo inhibition of the ethanol-oxidizing activity of ADH family by aspirin using steady-state inhibition equations.
Recombinant human class I ADHs exhibited lower Km as well as higher catalytic efficiency ( Vmax/Km ) than did the class II and class IV enzymes. Recombinant class I γ1-ADH and γ2-ADH showed negative cooperativity for ethanol oxidation with Hill coefficients (h) 0.607 and 0.401, respectively. Recombinant class III χ-ADH displayed extremely low Km for formaldehyde, 0.794 μM, in the presence of glutathione, indicating that it acts as a glutathione-dependent formaldehyde dehydrogenase.
Two inhibition patterns were found for ethanol oxidation by aspirin in the human ADH family. Competitive pattern was obtained for class I α-ADH with slope inhibition constant ( Kis ) 0.448 mM , class I β1-ADH ( Kis = 24.9 mM ), class II π-ADH ( Kis = 0.142 mM ), and class IV μ-ADH ( Kis = 6.36 mM ). Among them the class II enzyme exhibited the lowest inhibition constant. Noncompetitive pattern was obtained for class I β2-ADH with slope inhibition constant ( Kis ) 4.73 mM and intercept inhibition constant ( Kii ) 10.9 mM, and for class I β3-ADH ( Kis = 5.10 mM; Kii = 4.93 mM ).
High-Km hepatic class II π-ADH and gastric class IV μ-ADH can contribute effectively to first-pass metabolism of ethanol. Simulation studies using the steady-state inhibition equation with the determined kinetic parameters indicate that at physiologically attainable concentrations of aspirin, oxidation of ethanol by π-ADH or μ-ADH are inhibited most strongly. The results suggest that aspirin may affect first-pass metabolism and hence bioavailability of ethanol.
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