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Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects.
Biochemistry. 2002 Aug 06; 41(31):10158-65.B

Abstract

Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O. Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively. (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1. Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O.

Authors+Show Affiliations

Division of Biochemical Engineering, Institute of Food Technology, University of Agricultural Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria.No affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

12146981

Citation

Klimacek, Mario, and Bernd Nidetzky. "Examining the Relative Timing of Hydrogen Abstraction Steps During NAD(+)-dependent Oxidation of Secondary Alcohols Catalyzed By Long-chain D-mannitol Dehydrogenase From Pseudomonas Fluorescens Using pH and Kinetic Isotope Effects." Biochemistry, vol. 41, no. 31, 2002, pp. 10158-65.
Klimacek M, Nidetzky B. Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects. Biochemistry. 2002;41(31):10158-65.
Klimacek, M., & Nidetzky, B. (2002). Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects. Biochemistry, 41(31), 10158-65.
Klimacek M, Nidetzky B. Examining the Relative Timing of Hydrogen Abstraction Steps During NAD(+)-dependent Oxidation of Secondary Alcohols Catalyzed By Long-chain D-mannitol Dehydrogenase From Pseudomonas Fluorescens Using pH and Kinetic Isotope Effects. Biochemistry. 2002 Aug 6;41(31):10158-65. PubMed PMID: 12146981.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain D-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects. AU - Klimacek,Mario, AU - Nidetzky,Bernd, PY - 2002/7/31/pubmed PY - 2002/9/6/medline PY - 2002/7/31/entrez SP - 10158 EP - 65 JF - Biochemistry JO - Biochemistry VL - 41 IS - 31 N2 - Mannitol dehydrogenases (MDH) are a family of Zn(2+)-independent long-chain alcohol dehydrogenases that catalyze the regiospecific NAD(+)-dependent oxidation of a secondary alcohol group in polyol substrates. pH and primary deuterium kinetic isotope effects on kinetic parameters for reaction of recombinant MDH from Pseudomonas fluorescens with D-mannitol have been measured in H(2)O and D(2)O at 25 degrees C and used to determine the relative timing of C-H and O-H bond cleavage steps during alcohol conversion. The enzymatic rates decreased at low pH; apparent pK values for log(k(cat)/K(mannitol)) and log k(cat) were 9.2 and 7.7 in H(2)O, respectively, and both were shifted by +0.4 pH units in D(2)O. Proton inventory plots for k(cat) and k(cat)/K(mannitol) were determined at pL 10.0 using protio or deuterio alcohol and were linear at the 95% confidence level. They revealed the independence of primary deuterium isotope effects on the atom fraction of deuterium in a mixed H(2)O-D(2)O solvent and yielded single-site transition-state fractionation factors of 0.43 +/- 0.05 and 0.47 +/- 0.01 for k(cat)/K(mannitol) and k(cat), respectively. (D)(k(cat)/K(mannitol)) was constant (1.80 +/- 0.20) in the pH range 6.0-9.5 and decreased at high pH to a limiting value of approximately 1. Measurement of (D)(k(cat)/K(fructose)) at pH 10.0 and 10.5 using NADH deuterium-labeled in the 4-pro-S position gave a value of 0.83, the equilibrium isotope effect on carbonyl group reduction. A mechanism of D-mannitol oxidation by MDH is supported by the data in which the partly rate-limiting transition state of hydride transfer is stabilized by a single solvation catalytic proton bridge. The chemical reaction involves a pH-dependent internal equilibrium which takes place prior to C-H bond cleavage and in which proton transfer from the reactive OH to the enzyme catalytic base may occur. Loss of a proton from the enzyme at high pH irreversibly locks the ternary complex with either alcohol or alkoxide bound in a conformation committed of undergoing NAD(+) reduction at a rate about 2.3-fold slower than the corresponding reaction rate of the protonated complex. Transient kinetic studies for D-mannitol oxidation at pH(D) 10.0 showed that the solvent isotope effect on steady-state turnover originates from a net rate constant of NADH release that is approximately 85% rate-limiting for k(cat) and 2-fold smaller in D(2)O than in H(2)O. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/12146981/Examining_the_relative_timing_of_hydrogen_abstraction_steps_during_NAD_+__dependent_oxidation_of_secondary_alcohols_catalyzed_by_long_chain_D_mannitol_dehydrogenase_from_Pseudomonas_fluorescens_using_pH_and_kinetic_isotope_effects_ L2 - https://dx.doi.org/10.1021/bi025517x DB - PRIME DP - Unbound Medicine ER -