Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase

Abstract

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/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 (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, 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 kcat/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 was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.

Figure 1. Participation of Glu¹⁵⁵ in Zn²⁺ co-ordination by human liver SDH

The figure was derived from the 2.0 Å crystal structure of enzyme bound with NADH and an inhibitor (PDB: 1PL6) [5]. Residues within a 4 Å distance to Zn²⁺ are depicted. Black and grey dashed lines indicate co-ordination to Zn²⁺ and hydrogen bonding respectively.

Figure 2. pL profiles of k_cat and k_cat/K_sorbitol for slSDH and of k_cat/K_sorbitol for wild-type and E154C mutant forms of GmXDH

● and ○ indicate measurements in ¹H₂O and ²H₂O solvent respectively. Error bars give the S.D. of the respective kinetic parameter, and lines are non-linear fits of the data with the appropriate equation (see Table 2). In (C), △ indicates measurements with deuterated buffers that had been prepared through ¹H → ²H exchange via freeze-drying. ▲ Shows corresponding measurements in ¹H₂O. Insets show double-log plots of the data to illustrate differences in the shape of the pL profiles at low pH, (C), sigmoidal; (D), non-sigmoidal.

Figure 3. Stopped-flow progress curves of formation of NADH and release of proton during NAD⁺-dependent oxidation of sorbitol catalysed by slSDH

Traces with the higher signal-to-noise ratio are proton release measurements. Non-linear fits of the individual progress curves with Eqn (4) are shown as grey lines. Controls in which sorbitol was lacking and no reaction took place are also shown.

Figure 4. Alternative proton shuttle pathways for SDH/XDH

The structure of human liver SDH is shown (PDB: 1PL6) [5]. Hydrogen-bonded networks connecting the active site with bulk water are indicated with arrows. Black and grey dashed lines indicate co-ordination to Zn²⁺ and hydrogen bonding respectively.

Scheme 1. Proposed participation of Glu¹⁵⁴ in the catalytic mechanism of SDH/XDH