title: Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme.
abstract: A controversy exists with respect to the mechanism of l-2-hydroxy acid oxidation by members of a family of FMN-dependent enzymes. A so-called carbanion mechanism was initially proposed, in which the active site histidine abstracts the substrate α-hydrogen as a proton, followed by electron transfer from the carbanion to the flavin. But an alternative mechanism was not incompatible with some results, a mechanism in which the active site histidine instead picks up the substrate hydroxyl proton and a hydride transfer occurs. Even though more recent experiments ruling out such a mechanism were published (Rao & Lederer (1999) Protein Science 7, 1531-1537), a few authors have subsequently interpreted their results with variant enzymes in terms of a hydride transfer. In the present work, we analyse the reactivity of trifluorolactate, a substrate analogue, with the flavocytochrome b2 (Fcb2) flavodehydrogenase domain, compared to its reactivity with an NAD-dependent lactate dehydrogenase (LDH), for which this compound is known to be an inhibitor (Pogolotti & Rupley (1973) Biochem. Biophys. Res. Commun, 55, 1214-1219). Indeed, electron attraction by the three fluorine atoms should make difficult the removal of the α-H as a hydride. We also analyse the reactivity of trifluoropyruvate with the FMN- and NAD-dependent enzymes. The results substantiate a different effect of the fluorine substituents on the two enzymes compared to their normal substrates. In the discussion we analyse the conclusions of recent papers advocating a hydride transfer mechanism for the family of l-2-hydroxy acid oxidizing FMN-dependent enzymes.
authors.affiliation: Laboratoire d'Enzymologie, UPR 9063, CNRS, 91198 Gif-sur-Yvette Cedex, France; Laboratoire de Chimie Physique, CNRS UMR 8000, Université Paris-Sud, Université Paris-Saclay, 91405 Orsay Cedex, France. Electronic address: florence.lederer@u-psud.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Lederer
authors.affiliation: Laboratoire d'Enzymologie, UPR 9063, CNRS, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Vignaud
authors.affiliation: Laboratoire d'Enzymologie, UPR 9063, CNRS, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Paul
authors.initials: P
authors.lastname: North
authors.affiliation: Laboratoire d'Enzymologie, UPR 9063, CNRS, 91198 Gif-sur-Yvette Cedex, France.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Bodevin
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D005486:Flavin Mononucleotide / Q000378:metabolism*; D054879:Halogenation; D006801:Humans; D006860:Hydrogen Bonding; D006885:Hydroxybutyrates / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D011485:Protein Binding; D000072417:Protein Domains; D011522:Protons; D019289:Pyruvic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D006885:Hydroxybutyrates; D011522:Protons; D011994:Recombinant Proteins; D019344:Lactic Acid; C002479:2-hydroxy-3-butynoic acid; D005486:Flavin Mononucleotide; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)
keywords: Carbanion; Flavocytochrome b(2); Hydride transfer; Lactate dehydrogenase; Trifluorolactate; Trifluoropyruvate