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On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase.
Biochem J. 2003 Oct 01; 375(Pt 1):141-9.BJ

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+Show Affiliations

Institute of Biotechnology, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

12826012

Citation

Klimacek, Mario, et al. "On the Role of Brønsted Catalysis in Pseudomonas Fluorescens Mannitol 2-dehydrogenase." The Biochemical Journal, vol. 375, no. Pt 1, 2003, pp. 141-9.
Klimacek M, Kavanagh KL, Wilson DK, et al. On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase. Biochem J. 2003;375(Pt 1):141-9.
Klimacek, M., Kavanagh, K. L., Wilson, D. K., & Nidetzky, B. (2003). On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase. The Biochemical Journal, 375(Pt 1), 141-9.
Klimacek M, et al. On the Role of Brønsted Catalysis in Pseudomonas Fluorescens Mannitol 2-dehydrogenase. Biochem J. 2003 Oct 1;375(Pt 1):141-9. PubMed PMID: 12826012.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase. AU - Klimacek,Mario, AU - Kavanagh,Kathryn L, AU - Wilson,David K, AU - Nidetzky,Bernd, PY - 2003/06/24/accepted PY - 2003/05/20/received PY - 2003/6/27/pubmed PY - 2003/10/21/medline PY - 2003/6/27/entrez SP - 141 EP - 9 JF - The Biochemical journal JO - Biochem. J. VL - 375 IS - Pt 1 N2 - 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+. SN - 1470-8728 UR - https://www.unboundmedicine.com/medline/citation/12826012/On_the_role_of_Brønsted_catalysis_in_Pseudomonas_fluorescens_mannitol_2_dehydrogenase_ L2 - https://portlandpress.com/biochemj/article-lookup/doi/10.1042/BJ20030733 DB - PRIME DP - Unbound Medicine ER -