首页>
外文期刊>The biochemical journal
>The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites
【24h】
The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites
pThe side chains of Asnsup191/sup and Asnsup300/sup constitute a characteristic structural motif of the active site of iPseudomonas fluorescens/i mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as ~ 5×10sup2/sup ssup?1/sup and ~ 1.5×10sup3/sup ssup?1/sup in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by ≥1.2 pH units, in the kinetic piK/i of the catalytic acid-base Lyssup295/sup in the enzyme–NADsup+/sup–mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is ‘activated’ for chemical conversion through its precise alignment with the unprotonated side chain of Lyssup295/sup (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asnsup191/sup and Asnsup300/sup (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide ~ 40 kJ/mol of electrostatic stabilization./p
展开▼
