Examining the relative timing of hydrogen abstraction steps during NAD+-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects

Mannitol dehydrogenases (MDH) are a family of Zn2+-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H2O and D2O at 25 degreesC and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K-mannitol) and log k(cat) were 9.2 and 7.7 in H2O, respectively, and both were shifted by +0.4 pH units in D2O. Proton inventory plots for k(cat) and k(cat)/K-mannitol were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H2O-D2O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K-mannitol, and k(cat), respectively. 1(k(cat)/K-mannitol) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of Measurement of (D)(k(cat)/K-fructose) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism Of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximate to85% rate-limiting for k(cat) and 2-fold smaller in D2O than in H2O.