Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu-154->Cys forms of yeast xylitol dehydrogenase

Mario Klimacek , Heidemarie Hellmer , Bernd Nidetzky (1)

1 TU Graz - Graz University of Technology [Graz]

Coordination of catalytic Zn 2+} in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase (MDR) superfamily involves direct or water-mediated interactions from a Glu residue, which substitutes a homologous Cys ligand in alcohol dehydrogenases of the yeast and liver type. Glu-154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (GmXDH) was mutated into Cys (E154C) to revert this replacement. In spite of their variable Zn 2+} content (0.10 - 0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn 2+} centre activity (k cat}) of 1.19±0.03 s -1} and did not require exogenous Zn 2+} for activity or stability. E154C retained 0.019±0.003% and 0.74±0.03% of wild-type catalytic efficiency (k cat}/K sorbitol}; = 7800±700 M -1}s -1}) and k cat} (= 161±4 s -1}) for NAD +}-dependent oxidation of sorbitol at 25 °C, respectively. The pH profile of (k cat}/K sorbitol}) for E154C decreased below an apparent pK of 9.1±0.3, reflecting a shift in pK by about +1.7 - 1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (slSDH). The difference in pK for profiles determined in 1}H 2}O and 2}H 2}O solvent was similar and unusually small for all three enzymes (approx. +0.2 log units), suggesting that observed pK in binary enzyme-NAD+ complexes could be due to zinc 2+}-bound water. Under conditions eliminating different pH dependencies, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7±0.2 (E154C: 1.7±0.1) and 1.9±0.3 (E154C: 2.4±0.2) on k cat}/K sorbitol} respectively. Transient kinetic studies of NAD +} reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687±12 s -1} in the pre-steady state, which features a turnover of 0.9±0.1 enzyme equivalents as NADH produced with a rate constant of 409±3 s -1}. The results support an auxiliary participation of Glu-154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.

Mots clés: Life Sciences

Mario Klimacek, Heidemarie Hellmer, Bernd Nidetzky. Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu-154->Cys forms of yeast xylitol dehydrogenase. Biochemical Journal, 2007, 404 (3), pp.421-429. ⟨10.1042/BJ20061384⟩. ⟨hal-00478660⟩