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Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.
Biochemistry. 1999 Aug 10; 38(32):10489-98.B

Abstract

To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of </=1 above pH 9.6, corresponding to the pK of 9.34 observed in the pH profile of k(cat)/K(fructose). Therefore, hydride transfer is not pH-dependent, and D-fructose is not sticky at pH 7.0. A comparison of the kinetic data of MDH and mammalian sorbitol dehydrogenase, presumably involved in detoxification metabolism, is used to point out a physiological function of MDH in the oxidation of D-mannitol with high specificity and fluxional efficiency under prevailing reaction conditions in vivo.

Authors+Show Affiliations

Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien (BOKU), Austria.No affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10441145

Citation

Slatner, M, et al. "Kinetic Study of the Catalytic Mechanism of Mannitol Dehydrogenase From Pseudomonas Fluorescens." Biochemistry, vol. 38, no. 32, 1999, pp. 10489-98.
Slatner M, Nidetzky B, Kulbe KD. Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens. Biochemistry. 1999;38(32):10489-98.
Slatner, M., Nidetzky, B., & Kulbe, K. D. (1999). Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens. Biochemistry, 38(32), 10489-98.
Slatner M, Nidetzky B, Kulbe KD. Kinetic Study of the Catalytic Mechanism of Mannitol Dehydrogenase From Pseudomonas Fluorescens. Biochemistry. 1999 Aug 10;38(32):10489-98. PubMed PMID: 10441145.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens. AU - Slatner,M, AU - Nidetzky,B, AU - Kulbe,K D, PY - 1999/8/11/pubmed PY - 1999/8/11/medline PY - 1999/8/11/entrez SP - 10489 EP - 98 JF - Biochemistry JO - Biochemistry VL - 38 IS - 32 N2 - To characterize catalysis by NAD-dependent long-chain mannitol 2-dehydrogenases (MDHs), the recombinant wild-type MDH from Pseudomonas fluorescens was overexpressed in Escherichia coli and purified. The enzyme is a functional monomer of 54 kDa, which does not contain Zn(2+) and has B-type stereospecificity with respect to hydride transfer from NADH. Analysis of initial velocity patterns together with product and substrate inhibition patterns and comparison of primary deuterium isotope effects on the apparent kinetic parameters, (D)k(cat), (D)(k(cat)/K(NADH)), and (D)(k(cat)/K(fructose)), show that MDH has an ordered kinetic mechanism at pH 8.2 in which NADH adds before D-fructose, and D-mannitol and NAD are released in that order. Isomerization of E-NAD to a form which interacts with D-mannitol nonproductively or dissociation of NAD from the binary complex after isomerization is the slowest step (>/=110 s(-)(1)) in D-fructose reduction at pH 8.2. Release of NADH from E-NADH (32 s(-)(1)) is the major rate-limiting step in mannitol oxidation at this pH. At the pH optimum for D-fructose reduction (pH 7.0), the rate of hydride transfer contributes significantly to rate limitation of the catalytic cascade and the overall reaction. (D)(k(cat)/K(fructose)) decreases from 2.57 at pH 7.0 to a value of </=1 above pH 9.6, corresponding to the pK of 9.34 observed in the pH profile of k(cat)/K(fructose). Therefore, hydride transfer is not pH-dependent, and D-fructose is not sticky at pH 7.0. A comparison of the kinetic data of MDH and mammalian sorbitol dehydrogenase, presumably involved in detoxification metabolism, is used to point out a physiological function of MDH in the oxidation of D-mannitol with high specificity and fluxional efficiency under prevailing reaction conditions in vivo. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/10441145/Kinetic_study_of_the_catalytic_mechanism_of_mannitol_dehydrogenase_from_Pseudomonas_fluorescens_ L2 - https://dx.doi.org/10.1021/bi990327g DB - PRIME DP - Unbound Medicine ER -