Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxylase: stereochemistry, isotope effects, and kinetic mechanism
A neutral flavin semiquinone species was formed upon photoreduction of Pseudomonas cepacia 3-hydroxybenzoate 6-hydroxlase whereas no flavin radical was detected by anaerobic reduction with NADH in the presence of m-hydroxybenzoate. In the latter case, the formation of flavin semiquinone is apparently thermodynamically unfavorable. A stereospecificity for the abstraction of the 4R-position hydrogen of NADH has been demonstrated for this hydroxylase. Deuterium and tritium isotope effects were observed with (4R)-(4-/sup 2/H)NADH and (4R)-(4-/sup 3/H)NADH as substrates. The /sub D/V effect indicates the existence of at least one slow step after the isotope-sensitive enzyme reduction by dihydropyridine nucleotide. A minimal kinetic mechanism has been deduced on the basis of initial velocity measurements and studies on deuterium and tritium isotope effects. Following this scheme, m-hydroxybenzoate and NADH bind to the hydroxylase in a random sequence. The flavohydroxylase is reduced by NADH, and NAD/sup +/ is released. Oxygen subsequently binds to and reacts with the reduced flavohydroxylase-m-hydroxybenzoate complex. Following the formation and release of water and gentisate, the oxidized holoenzyme is regenerated. The enzyme has a small preference for the release of NADH over m-hydroxybenzoate from the enzyme-substrates ternary complex.
- Research Organization:
- Univ. of Houston-University Park, TX
- OSTI ID:
- 6793120
- Journal Information:
- Biochemistry; (United States), Vol. 26:4
- Country of Publication:
- United States
- Language:
- English
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HYDROXYLASES
ISOTOPE EFFECTS
STEREOCHEMISTRY
PSEUDOMONAS
BIOCHEMICAL REACTION KINETICS
BENZOHYDROXAMIC ACID
DEUTERIUM COMPOUNDS
NADH2
TRITIUM COMPOUNDS
AMINES
BACTERIA
COENZYMES
ENZYMES
HYDROGEN COMPOUNDS
HYDROXAMIC ACIDS
HYDROXY COMPOUNDS
KINETICS
LABELLED COMPOUNDS
MICROORGANISMS
NUCLEOTIDES
ORGANIC COMPOUNDS
OXIDOREDUCTASES
REACTION KINETICS
550201* - Biochemistry- Tracer Techniques