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 meas ures 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 DeltaV (double dagger) = -9.7 +/- 1.0 mL/mol. Pressures above 1.5 kbar decrease ca pture, 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 t ightly bound reactant-state complex, E*-NAD(+), with DeltaV* = +83.4 +/- 5. 2 mL/mol. A comparison of the effects of pressure on the oxidation of norma l and deuteroformate shows that the entire isotope effect on hydride transf er, 2.73 +/- 0.20, arises solely from transition-state phenomena, as was al so 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 mi xture of both reactant-state and transition-state phenomena. Moreover, pres sure increases the primary intrinsic deuterium isotope effect, the opposite of what was observed with yeast alcohol dehydrogense. The lack of a decrea se in the isotope effect is also contrary to empirical precedents from chem ical reactions suspected of tunneling and to theoretical constructs of vibr ationally enhanced tunneling in enzymatic reactions. Hence, this new experi mental 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.