Abstract
D-amino acid oxidases (DAAO) are stereospecific enzymes which are generally almost inactive towards L-enantiomer in neutral solution when L-, D-amino acids are supplied as substrates. In this paper, the D-amino acid oxidase can catalytic oxidize L-amino acids by modulating pH of aqueous solution. With L-Pro as substrate, the catalytic rate (kcat) and the affinity (Km) of DAAO were 6.71 s−1 and 33 mM at pH 8.0, respectively, suggesting that optimal pH condition enhanced the activity of DAAO towards L-Pro. Similar results were obtained when L-Ala (pH 9.8), L-Arg (pH 6.5), L-Phe (pH 9.0), L-Thr (pH 9.4), and L-Val (pH 8.5) were catalyzed by DAAO at various pH values. The racemization of the L-amino acids was not found by capillary electrophoresis analysis during oxidation, and quantification analysis of L-amino acids before and after catalytic reaction was performed, which confirmed that the modulation of enantioselectivity of DAAO resulted from the oxidation of L-amino acids rather than D-amino acids by changing pH. A mechanistic model was proposed to explain enhanced activity of DAAO towards L-amino acids under optimal pH condition.
Highlights
D-amino acid oxidase (DAAO) is the prototype of the FAD-dependent flavoprotein, which catalyzes oxidative deamination of a majority of D-amino acids rather than L-amino acids, producing the corresponding α-keto acid and ammonia[1,2,3]
Molecular modeling and optimization suggested that the enhanced catalytic activity of D-amino acid oxidases (DAAO) towards L-Ala at pH 9.8 may be due to the easy transpositions of amino group and methyl group of L-Ala binding to DAAO when positively charged amino group was changed into electrically neutral amino group under alkaline conditions
Such a study is necessary to understand the structure of active center, especially the state of charge, contributing alteration of stereospecificity of DAAO, which may provide a positive guidance for asymmetric catalytic oxidation of chiral amino acids by DAAO in aqueous solution
Summary
D-amino acid oxidase (DAAO) is the prototype of the FAD-dependent flavoprotein, which catalyzes oxidative deamination of a majority of D-amino acids rather than L-amino acids, producing the corresponding α-keto acid and ammonia[1,2,3]. Tawaki et al.[25] found a complete reversal of enantioselectivity of the transesterification catalyzed by Aspergillus oryzae protease with 18 anhydrous solvents These studies have clearly shown that the changes of enzyme enantioselectivity can be achieved by using organic solvents, whereas little attention has been focused on the exploitation of aqueous solution, which is more important for the living systems. Molecular modeling and optimization suggested that the enhanced catalytic activity of DAAO towards L-Ala at pH 9.8 may be due to the easy transpositions of amino group and methyl group of L-Ala binding to DAAO when positively charged amino group was changed into electrically neutral amino group under alkaline conditions Such a study is necessary to understand the structure of active center, especially the state of charge, contributing alteration of stereospecificity of DAAO, which may provide a positive guidance for asymmetric catalytic oxidation of chiral amino acids by DAAO in aqueous solution
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