Effects of pressure on deuterium isotope effects of yeast alcohol dehydrogenase using alternative substrates

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 ΔV‡ = −30 mL mol−1 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 mol−1. 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 mol−1 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.