Kinetic mechanism and nucleotide specificity of NADH peroxidase
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.
- Research Organization:
- Albert Einstein College of Medicine, Bronx, NY (USA)
- OSTI ID:
- 5272284
- Journal Information:
- Arch. Biochem. Biophys.; (United States), Vol. 260:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
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