title: Revisitation of the βCl-elimination reaction of D-amino acid oxidase: new interpretation of the reaction that sparked flavoprotein dehydrogenation mechanisms.
abstract: D-amino acid oxidase (DAAO) from pig has been reported to catalyze the β-elimination of Cl(-) from βCl-D-alanine via abstraction of the substrate α-H as H(+) ("carbanion mechanism") (Walsh, C. T., Schonbrunn, A., and Abeles, R. H. (1971) J. Biol. Chem. 246, 6855-6866). In view of the fundamental mechanistic importance of this reaction and of the recent reinterpretation of the DAAO dehydrogenation step as occurring via a hydride mechanism, we reinvestigated the elimination reaction using yeast DAAO. That enzyme catalyzes the same reactions as the pig enzyme but with a much higher efficiency and a substantially different kinetic behavior. The reaction is initiated by a very rapid and fully reversible dehydrogenation step. This leads to an equilibrium (k(on) ≈ k(reverse)) between the complexes of oxidized enzyme-βCl-D-alanine and reduced enzyme-βCl-iminopyruvate. In the presence of O(2) the latter complex can partition between an oxidative half-reaction and elimination of Cl(-), which proceeds at a rate of ≈50 s(-1). This step forms a complex between oxidized enzyme and enamine that is characterized by a charge transfer absorption (which describes its rates of formation and decay). A minimal scheme that lists relevant steps of the reductive and oxidative half-reactions and elimination pathways along with the estimate of the corresponding rate constants is presented. β-Elimination of Cl(-) is proposed to originate at the locus of the enzyme-βCl-iminopyruvate complex. A chemical mechanism that can account for elimination is discussed in detail.
authors.affiliation: Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell'Insubria, via J. H. Dunant 3, 21100 Varese, Italy.
authors.forename: Sandro
authors.initials: S
authors.lastname: Ghisla
authors.forename: Loredano
authors.initials: L
authors.lastname: Pollegioni
authors.forename: Gianluca
authors.initials: G
authors.lastname: Molla
mesh_terms: D000332:Aerobiosis; D000409:Alanine / Q000737:chemistry* / Q000378:metabolism*; D000693:Anaerobiosis; D000818:Animals; D002713:Chlorine / Q000737:chemistry*; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism; D005420:Flavoproteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006865:Hydrogenation; D007554:Isotopes; D007700:Kinetics; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology; D013552:Swine
chemical_list: D005415:Flavins; D005420:Flavoproteins; D007554:Isotopes; D002713:Chlorine; D019289:Pyruvic Acid; D003605:D-Amino-Acid Oxidase; D000409:Alanine; D010100:Oxygen