skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Kinetic mechanism and nucleotide specificity of NADH peroxidase

Abstract

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between (/sup 14/C)NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilicmore » attack by enzymatic dithiols.« less

Authors:
;
Publication Date:
Research Org.:
Albert Einstein College of Medicine, Bronx, NY (USA)
OSTI Identifier:
5272284
Resource Type:
Journal Article
Journal Name:
Arch. Biochem. Biophys.; (United States)
Additional Journal Information:
Journal Volume: 260:2
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; PEROXIDASES; BIOCHEMICAL REACTION KINETICS; SPECIFICITY; CARBON 14 COMPOUNDS; DEUTERIUM; HYDROGEN PEROXIDE; ION EXCHANGE; ISOTOPE EFFECTS; NAD; NADP; STREPTOCOCCUS; TRACER TECHNIQUES; TRITIUM COMPOUNDS; WATER; BACTERIA; COENZYMES; ENZYMES; HYDROGEN COMPOUNDS; HYDROGEN ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; KINETICS; LABELLED COMPOUNDS; LIGHT NUCLEI; MICROORGANISMS; NUCLEI; NUCLEOTIDES; ODD-ODD NUCLEI; ORGANIC COMPOUNDS; OXIDOREDUCTASES; OXYGEN COMPOUNDS; PEROXIDES; REACTION KINETICS; STABLE ISOTOPES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Stoll, V S, and Blanchard, J S. Kinetic mechanism and nucleotide specificity of NADH peroxidase. United States: N. p., 1988. Web. doi:10.1016/0003-9861(88)90505-X.
Stoll, V S, & Blanchard, J S. Kinetic mechanism and nucleotide specificity of NADH peroxidase. United States. https://doi.org/10.1016/0003-9861(88)90505-X
Stoll, V S, and Blanchard, J S. 1988. "Kinetic mechanism and nucleotide specificity of NADH peroxidase". United States. https://doi.org/10.1016/0003-9861(88)90505-X.
@article{osti_5272284,
title = {Kinetic mechanism and nucleotide specificity of NADH peroxidase},
author = {Stoll, V S and Blanchard, J S},
abstractNote = {NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between (/sup 14/C)NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.},
doi = {10.1016/0003-9861(88)90505-X},
url = {https://www.osti.gov/biblio/5272284}, journal = {Arch. Biochem. Biophys.; (United States)},
number = ,
volume = 260:2,
place = {United States},
year = {Mon Feb 01 00:00:00 EST 1988},
month = {Mon Feb 01 00:00:00 EST 1988}
}