title: On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase.
abstract: X-ray structure of the Pseudomonas fluorescens mannitol 2-dehydrogenase ternary complex with NAD+ and D-mannitol suggests that Lys-295 provides catalytic base assistance to secondary alcohol group oxidation. We have replaced Lys-295 by site-directed mutagenesis with alanine or methionine and evaluated the catalytic significance of side-chain substitution by kinetic analysis of restoration of activity with external amines, and from pH and solvent isotope effects on the reaction catalysed by K295A (Lys-295-->Ala mutant). K295A and K295M (Lys-295-->Met mutants) show 3x10(4)- and 2x10(6)-fold lower turnover numbers respectively for D-mannitol oxidation (kcatO) at pH 10.0 than the wild-type. The second-order rate constant for non-covalent rescue of activity (kB) by free methylamine base is 31 M(-1) x s(-1) for K295A, but only 0.021 M(-1) x s(-1) for K295M. A Brønsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope beta=0.66+/-0.16; r2=0.99) for K295A-catalysed D-mannitol oxidation at pH 10.0. The kcatO values of K295A in H2O and 2H2O are linearly dependent on [OL-] in the pL range 7.5-10.5 (where L is 1H or 2H). The solvent isotope effect on kcatO is 0.69. The time course of D-fructose reduction by K295A at pH 8.2 displays a pre-steady-state burst of NADH consumption. These data support a mechanism in which the epsilon -NH2 group of Lys-295 participates in an obligatory pH-dependent, pre-catalytic equilibrium which may control alcohol/alkoxide equilibration of enzyme-bound D-mannitol and activates the C2 atom for subsequent catalytic oxidation by NAD+.
authors.affiliation: Institute of Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
authors.forename: Mario
authors.initials: M
authors.lastname: Klimacek
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Kavanagh
authors.forename: David K
authors.initials: DK
authors.lastname: Wilson
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000588:Amines / Q000737:chemistry; D002384:Catalysis; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008353:Mannitol / Q000737:chemistry / Q000378:metabolism; D008354:Mannitol Dehydrogenases; D008956:Models, Chemical; D009154:Mutation; D010084:Oxidation-Reduction; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D012997:Solvents / Q000737:chemistry
chemical_list: D000588:Amines; D012997:Solvents; D008353:Mannitol; D003903:Deuterium; D000429:Alcohol Oxidoreductases; D008354:Mannitol Dehydrogenases; C116789:mannitol 2-dehydrogenase (NADP); D008239:Lysine