On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase

Mario Klimacek, Kathryn L. Kavanagh, David K. Wilson, Bernd Nidetzky

Research output: Contribution to journalArticle

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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 3 × 10 4- and 2 × 106-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 · s -1 for K295A, but only 0.021 M-1 · s-1 for K295M. A Brønsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope β= 0.66 ± 0.16; r2 = 0.99) for K295A-catalysed D-mannitol oxidation at pH 10.0. The kcatO values of K295A in H 2O 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 ε-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+.

Original languageEnglish (US)
Pages (from-to)141-149
Number of pages9
JournalBiochemical Journal
Volume375
Issue number1
DOIs
StatePublished - Oct 1 2003

Fingerprint

Mannitol Dehydrogenases
Pseudomonas fluorescens
Mannitol
Catalysis
NAD
Isotopes
Oxidation
Amines
Alcohols
Mutagenesis
Catalytic oxidation
Fructose
Alanine
Methionine
Restoration
Rate constants
Site-Directed Mutagenesis
Substitution reactions
X rays
Atoms

Keywords

  • Alkoxide mechanism
  • Catalytic base
  • Long-chain dehydrogenases
  • Stepwise hydrogen transfer

ASJC Scopus subject areas

  • Biochemistry

Cite this

On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase. / Klimacek, Mario; Kavanagh, Kathryn L.; Wilson, David K.; Nidetzky, Bernd.

In: Biochemical Journal, Vol. 375, No. 1, 01.10.2003, p. 141-149.

Research output: Contribution to journalArticle

Klimacek, M, Kavanagh, KL, Wilson, DK & Nidetzky, B 2003, 'On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase', Biochemical Journal, vol. 375, no. 1, pp. 141-149. https://doi.org/10.1042/BJ20030733
Klimacek, Mario ; Kavanagh, Kathryn L. ; Wilson, David K. ; Nidetzky, Bernd. / On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase. In: Biochemical Journal. 2003 ; Vol. 375, No. 1. pp. 141-149.
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AB - 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 3 × 10 4- and 2 × 106-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 · s -1 for K295A, but only 0.021 M-1 · s-1 for K295M. A Brønsted relationship of log kB (corrected for molecular size effects) and pKa of the external amine is linear (slope β= 0.66 ± 0.16; r2 = 0.99) for K295A-catalysed D-mannitol oxidation at pH 10.0. The kcatO values of K295A in H 2O 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 ε-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+.

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