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.
Highlights
D-Amino acid oxidase (DAAO) from pig has been reported to catalyze the -elimination of Cl؊ from Cl-D-alanine via abstraction of the substrate ␣-H as H؉ (“carbanion mechanism”)
A chemical mechanism that can account for elimination is discussed in detail. In their seminal paper that appeared in this journal in 1971, Walsh et al [1] reported that upon incubation with D-amino acid oxidase (DAAO)2 (EC 1.4.3.3) Cl-D-alanine (Cl-D-Ala) eliminates ClϪ, which was later confirmed by others [2,3,4]
The structures of complexes of Rhodotorula gracilis D-amino acid oxidase (RgDAAO) with D-alanine or D-CF3-alanine show that the substrate ␣C-H points directly toward the flavin N(5) and is poised for hydride transfer [13]
Summary
Enzymes and Buffers—D-Amino acid oxidase from R. gracilis was produced and purified from recombinant BL21(DE3)pLysS E. coli cells carrying the pT7-DAAO expression plasmid as reported by Molla et al [17]. Absorption Measurements and Pyruvate Molar Extinction Coefficient—Enzyme concentration is indicated in terms of flavin content using an ⑀455 nm ϭ 12,600 MϪ1 cmϪ1 [16, 17]. The pyruvate molar extinction coefficient at 320 nm was determined as 20 Ϯ 1 MϪ1 cmϪ1 by linear fitting of the absorbance values of samples containing increasing, known concentrations of pyruvate in composite buffer, pH 7.0, at 25 °C. Samples were Ϸ0.5 ml in 5-mm tubes, and measurements were performed at 28 °C; 8 –16 pulses were recorded and averaged for Fourier transformation. Rapid Reaction (Stopped Flow) Measurements—Rapid reaction measurements were carried out in composite buffer at 25 °C using a stopped-flow spectrophotometer equipped with a thermostat and a diode array detector (J&M Analytische Messund Regeltechnik GmbH) as detailed (18 –20). HPLC Analysis—HPLC was performed as described earlier [21]
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