PH-Dependent Enantioselectivity of D-amino Acid Oxidase in Aqueous Solution

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 (k _cat) and the affinity (K _m) 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.

Figure 1

CE analysis of solution of derivatized L-Pro (a) and the mixture solution of L-Pro and D-Pro after derivatization (b) after 30 min incubation at pH 8.0 at 37 °C.

Figure 2

Graphical presentation of the active site cavity of DAAO in complex with both enantiomers of Ala at different pH. The active site residues, the L-Ala and D-Ala molecule were shown in stick representation colored in green (carbon atoms), blue (nitrogen atoms) and red (oxygen atoms). The molecular modeling displayed DAAO in complex with D-Ala at pH 7.0 (a), DAAO in complex with L-Ala at pH 7.0 (b), DAAO in complex with D-Ala at pH 9.8 (c), DAAO in complex with L-Ala at pH 9.8 (d), respectively.