Effect of Pressure on Deuterium Isotope Effects of Formate Dehydrogenase

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Abstract
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) Biochemistry39, 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 observed previously with yeast alcohol dehydrogense. In contrast, normal primary isotope effects arise solely from different zero-point energies in reactant states, and those that express hydrogen tunneling arise from a mixture of both reactant-state and transition-state phenomena. Moreover, pressure increases the primary intrinsic deuterium isotope effect, the opposite of what was observed with yeast alcohol dehydrogense. The lack of a decrease in the isotope effect is also contrary to empirical precedents from chemical reactions suspected of tunneling and to theoretical constructs of vibrationally enhanced tunneling in enzymatic reactions. Hence, this new experimental design penetrates transition states of enzymatic catalysis as never before, reveals the presence of phenomena foreign to chemical kinetics, and calls for explanations of how enzymes work beyond the tenants of physical organic chemistry.