The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilisation in alcohol dehydrogenase active sites
Résumé
The side chains of Asn-191 and Asn-300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens 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 asparagines by Ala. The rate constants for the overall hydride transfer to and from C2 of mannitol which were estimated as ~5 × 102 s-1 and ∼1.5 × 103 s-1 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 Asn residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by ≥ 1.2 pH units, in the kinetic pK of the catalytic acid-base Lys-295 in the enzyme-NAD+-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 Lys-295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn-191 and Asn-300 (fructose reduction). In the subsequent hydride transfer step, the two Asn residues provide ~40 kJ/mol of electrostatic stabilisation.
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