Temperature-Independent Kinetic Isotope Effects as Evidence for a Marcus-like Model of Hydride Tunneling in Phosphite Dehydrogenase

Abstract

Phosphite dehydrogenase catalyzes the transfer of a hydride from phosphite to NAD+, producing phosphate and NADH. We have evaluated the role of hydride tunneling in a thermostable variant of this enzyme (17X-PTDH) by measuring the temperature dependence of the primary 2H kinetic isotope effects (KIEs) between 5 and 45 °C. Pre-steady-state kinetic measurements were used to demonstrate that the hydride transfer is rate-determining across this temperature range and that the observed KIEs are equal to the intrinsic isotope effect on the chemical step. The KIEs on the pre-exponential factor (A H/A D) and the activation energy (ΔE a) were 1.6 ± 0.1 and 0.21 ± 0.05 kcal/mol, respectively, suggesting that 17X-PTDH facilitates extensive tunneling of both isotopes via a Marcus-like model. Site-directed mutagenesis was used to evaluate the role of an active site threonine (Thr104) found on the back face of the nicotinamide in promoting the close packing of the substrates. In mutants with reduced steric bulk at this position, values of A H/A D and ΔE a fall within the range describing semiclassical “over the barrier” reactivity, suggesting that Thr104 acts as a steric backstop to promote tunneling in 17X-PTDH. Whereas hydrogen tunneling is now a widely appreciated feature of C–H activating enzymes, these observations with a P–H activating system are consistent with the proposal that tunneling is likely to be a common feature on all enzymes that catalyze hydrogen transfers.

Substitutions of Amino Acid Residues in the Substrate Binding Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on the Structures but Significantly Affect Catalysis of Hydrogen Transfer

“…Nevertheless, the V203A substitution might allow the nicotinamide ring to fluctuate and affect the puckering during catalysis, which could be reflected in the diminished hydrogen tunneling, but the geometry of the ring and the temperature factors are very similar to those in the other enzymes (Tables and ). However, a new water molecule is hydrogen bonded to the guanidinium NH2 of Arg-369 and the phosphate O1N of NAD and also interacts with C6N of the nicotinamide ring. , Oxygens of highly conserved residues interact with the nicotinamide ring, Val-292 O to C2N and Thr-178 OG2 to C4N, in the ADH structures reported here and might also affect the chemistry of the nicotinamide ring. , It is interesting that the substitution of the homologous threonine (Thr-104) in phosphite dehydrogenase affects catalysis and hydrogen tunneling …”

“…3,70 It is interesting that the substitution of the homologous threonine (Thr-104) in phosphite dehydrogenase affects catalysis and hydrogen tunneling. 71 Dynamics of Catalysis. According to models that explain the energetics of enzyme catalysis (described in textbooks), enzymes use the energy gained from substrate binding to orient the reactants, destabilize the ground state, and stabilize the transition state.…”

Substituent Effects on Temperature Dependence of Kinetic Isotope Effects in Hydride-Transfer Reactions of NADH/NAD+ Analogues in Solution: Reaction Center Rigidity Is the Key

“…In the past two decades, however, it has been frequently observed that KIEs (both small and large) are temperature independent (Δ E a ≈ 0) in the wild-type enzymes ( wt -enzymes) around physiological temperature conditions, but they become temperature dependent to various extents with enzyme variants (Δ E a > 0 or even above the semiclassical limit). − A few contemporary H-tunneling theories have been established or used to explain the unusually small Δ E a and its change relating to enzyme structures and further to attempt to provide information for the possible role of protein thermal motions in catalysis. ,,− One largely used is the vibration-assisted activated H-tunneling (VA-AHT) model, which could include the Marcus-like model and TS theory extension, both of which involve a full H-tunneling process. ,, These phenomenological models presume that heavy atom motions bring H-donor and -acceptor to a tunneling-ready-state (TRS) where the activated reactant and product moieties have matching energy, allowing H-tunneling to occur over a range of donor–acceptor distances (DADs) sampled by the constructive heavy atom vibrations. Within that model, KIE is a function of DAD TRS , and its temperature dependence is related to the density of DAD TRS distributions. , Therefore, the Δ E a ≈ 0 with wt -enzymes has been explained in terms of the well-organized reaction coordinate in which DAD TRS is short and the range of DAD TRS ’s sampled is narrow.…”

Examining the Mechanism of Phosphite Dehydrogenase with Quantum Mechanical/Molecular Mechanical Free Energy Simulations

“…These calculations neglect the effects of hydrogen tunneling, which was implicated by a recent analysis of the temperature dependence of the primary hydrogen/deuterium (H/D) kinetic isotope effects (KIEs) for this enzymatic reaction. 46 In this previous work, the experimental H/D KIE was found to increase from 2.23 to 2.30 as the temperature was increased from 5 to 45 °C. The theoretical analysis of such effects is challenging given the complexity of the system, the small temperature range studied, and the small change in the KIE with temperature.…”

“…These calculations neglect the effects of hydrogen tunneling, which was implicated by a recent analysis of the temperature dependence of the primary hydrogen/deuterium (H/D) kinetic isotope effects (KIEs) for this enzymatic reaction . In this previous work, the experimental H/D KIE was found to increase from 2.23 to 2.30 as the temperature was increased from 5 to 45 °C.…”