Substitution of Tyr254 with Phe at the active site of flavocytochrome b2: consequences on catalysis of lactate dehydrogenation
- INSERM U 25, CNRS UA 122, Hopital Necker, Paris (France)
A role for Tyr254 in L-lactate dehydrogenation catalyzed by flavocytochrome b2 has recently been proposed on the basis of the known active-site structure and of studies that had suggested a mechanism involving the initial formation of a lactate carbanion. This role is now examined after replacement of Tyr254 with phenylalanine. The kcat is decreased about 40-fold, Km for lactate appears unchanged, and the mainly rate-limiting step is still alpha-hydrogen abstraction, as judged from the steady-state deuterium isotope effect. Modeling studies with lactate introduced into the active site indicate two possible substrate conformations with different hydrogen-bonding partners for the substrate hydroxyl. If the hydrogen bond is formed with Tyr254, as was initially postulated, the mechanism must involve removal by His373 of the C2 hydrogen, with carbanion formation. If, in the absence of the Tyr254 phenol group, the hydrogen bond is formed with His373 N3, the substrate is positioned in such a way that the reaction must proceed by hydride transfer. Therefore the mechanism of the Y254F enzyme was investigated so as to distinguish between the two mechanistic possibilities. 2-Hydroxy-3-butynoate behaves with the mutant as a suicide reagent, as with the wild-type enzyme. Similarly, the mutant protein also catalyzes the reduction and the dehydrohalogenation of bromopyruvate under transhydrogenation conditions.
- OSTI ID:
- 6284270
- Journal Information:
- Biochemistry; (USA), Vol. 29:27; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
Similar Records
A bacterial flavin‐dependent oxidoreductase that captures carbon dioxide into biomass
Functional Annotation of Two New Carboxypeptidases from the Amidohydrolase Superfamily of Enzymes
Related Subjects
LACTATE DEHYDROGENASE
MOLECULAR BIOLOGY
TYROSINE
BIOLOGICAL FUNCTIONS
CATALYSIS
CYTOCHROMES
DEUTERIUM
ISOTOPE EFFECTS
LACTATES
RECEPTORS
SACCHAROMYCES CEREVISIAE
STEADY-STATE CONDITIONS
TRACER TECHNIQUES
AMINO ACIDS
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
ENZYMES
EUMYCOTA
FUNCTIONS
FUNGI
HEMIACETAL DEHYDROGENASES
HYDROGEN ISOTOPES
HYDROXY ACIDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MEMBRANE PROTEINS
MICROORGANISMS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PIGMENTS
PLANTS
PROTEINS
SACCHAROMYCES
STABLE ISOTOPES
YEASTS
550201* - Biochemistry- Tracer Techniques