Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9882436 | Archives of Biochemistry and Biophysics | 2005 | 6 Pages |
Abstract
Hydrostatic pressure causes biphasic effects on the oxidation of alcohols by yeast alcohol dehydrogenase as expressed on the kinetic parameter V/K which measures substrate capture. Moderate pressure increases capture by activating hydride transfer, whose transition-state must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme, with ÎVâ¡Â = â30 mL molâ1 for isopropanol. A comparison of these effects with those on the oxidation of deutero-isopropanol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 7.6 mL molâ1. The pressure data extrapolate and factor the kinetic isotope effect into a semi-classical reactant-state component, with a null value of kH/kD = 1, and a transition-state component of QH/QD = 4, suggestive of hydrogen tunneling. Pressures above 1.5 kbar decrease capture by favoring a minor conformation of enzyme which binds nicotinamide adenine dinucleotide (NAD+) less tightly. This inactive conformation has a smaller volume than active E-NAD+, with a difference of 74 mL molâ1 and an equilibrium constant of 93 between them, at one atmosphere of pressure. These results are virtually identical to those obtained with benzyl alcohol [Cho and Northrop, Biochemistry 39 (2000) 2406] and give credence to this method of analysis. Moreover, qualitatively similar results with greater pressure sensitivity but less precision are obtained using ethanol as a substrate, only with pressure driving the value of the isotope effect to a value less than Dk = 1.03 directly, without extrapolation. The ethanol data verify the most surprising finding of these studies, namely that the entire kinetic isotope effect arises from a transition-state phenomenon.
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Authors
Hyun Park, Gene Kidman, Dexter B. Northrop,