Kinetic mechanism and nucleotide specificity of NADH peroxidase

Journal Article · Mon Feb 01 04:00:00 EST 1988 · Arch. Biochem. Biophys.; (United States)

DOI:https://doi.org/10.1016/0003-9861(88)90505-X· OSTI ID:5272284

Stoll, V S; Blanchard, J S

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), Journal Name: Arch. Biochem. Biophys.; (United States) Vol. 260:2; ISSN ABBIA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
COENZYMES
DEUTERIUM
ENZYMES
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
HYDROGEN PEROXIDE
ION EXCHANGE
ISOTOPE APPLICATIONS
ISOTOPE EFFECTS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MICROORGANISMS
NAD
NADP
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
OXYGEN COMPOUNDS
PEROXIDASES
PEROXIDES
REACTION KINETICS
SPECIFICITY
STABLE ISOTOPES
STREPTOCOCCUS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
WATER