A theoretical analysis of the proton and hydride transfer in liver alcohol dehydrogenase (LADH)

The proton and hydride transfers in horse liver alcohol dehydrogenase (LADH) were studied with a potential surface obtained by use of the self-consistent-charge-density-functional-tight-binding (SCC-DFTB) QM/ MM method implemented in the CHARMM program; a correction for solvent shielding was introduced by use of a continuum model. The proton transfers were found to proceed in a virtually concerted fashion before the hydride transfer. The calculations also showed that a radical mechanism, suggested as a possibility in the literature for the H transfer between the substrate and NAD+, is very unlikely. The energetics of the reaction and pK(a)'s of residues involved in catalysis indicate that the chemical steps of LADH, as characterized by the calculated value of k(cat) are slow for a pH below 5.5, and the hydride transfer is hardly affected for pH between 5.5 and 8.1. These results are compared with the experimentally measured pH dependence of k(cat) for LADH, although a quantitative comparison is difficult because the chemical steps are only partially rate-limiting in the experiments. A perturbation analysis of the QM/MM energies suggest that a number of charged residues close to the active site (i.e., Asp 49, Glu 68, and Arg 369), as well as the phosphate groups of NAD+, make important contributions to the energetics of the proton and hydride transfer reactions; mutation experiments to test these predictions would be of interest. Ser 48 interacts with the substrate via a short hydrogen bond, which leads to an inverse solvent isotope effect, in accord with experiment. The overall calculated barrier and endothermicity and the effect of the double mutation (F93W,V203A) on the hydride transfer are in qualitative agreement with measurements; i.e., the hydride transfer barrier is higher in the mutant, presumably due mainly to the fact that the average distance between the donor and acceptor is larger. In accord with the study of Alhambra et al. (Alhambra, C.; Corchado, J. C.; Sanchez, M. L.; Gao, J.; Truhlar, D. G. J. Am. Chem. Soc. 2000, 122, 8197.), hydride tunneling was shown to be very important for the calculated magnitude of kinetic isotope effects and the Swain-Schaad exponents, although the absolute contribution of tunneling to the rate constant is calculated to be small (a factor of 2) at room temperature. It was shown that the secondary Swain-Schaad exponent can be affected by the variation in the position of the transition state upon secondary isotopic substitution. The exponent, therefore, does not necessarily reflect the magnitude of tunneling; i.e., one has to be cautious in using the secondary Swain-Schaad exponent to estimate the magnitude of tunneling. Equilibrium effects of protein "dynamics" on the hydride transfer in LADH were studied by potential of mean force calculations at different temperatures. To obtain a clearer description of the origin of the calculated variation, an analysis was made with the QM and MM atoms maintained at different temperatures by use of Nose-Hoover thermostats. It was found that the barrier is correlated primarily with the temperature of the QM region, while the temperature of the MM atoms has a larger effect on the exothermicity. The variation of the barrier with the structures accessible by molecular dynamics simulations was found to be small (< 1 kcal/mol). The effect arises mainly from the change in the position of residues that directly interact with the reacting groups, such as Ser 48.