Effect of pressure on deuterium isotope effects of formate dehydrogenase.

High pressure causes biphasic effects on the oxidation of formate by yeast formate dehydrogenase as expressed on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by accelerating hydride transfer. The transition state for hydride transfer has a smaller volume than the free formate plus the capturing form of enzyme, with ΔV‡ = −9.7 ± 1.0 mL/mol. Pressures above 1.5 kbar decrease capture, reminiscent of effects on the conformational change associated with the binding of nicotinamide adenine dinucleotide (NAD+) to yeast alcohol dehydrogenase [Northrop, D. B., and Y. K. Cho (2000) Biochemistry 39, 2406−2412]. The collision complex, E-NAD+, has a smaller volume than the more tightly bound reactant-state complex, E*-NAD+, with ΔV* = +83.4 ± 5.2 mL/mol. A comparison of the effects of pressure on the oxidation of normal and deuteroformate shows that the entire isotope effect on hydride transfer, 2.73 ± 0.20, arises solely from transition-state phenomena, as was also ...