L-Arabinonate → 2-dehydro-3-deoxy-D-arabinonate

Participants

Reaction Steps

D-Xylonate and L-arabinonate dehydratase can be assayed according to procedures described by Dahms. (Dahms, A. S.; Donald, A. Meth. Enzymol. 1982, 90, 302.; Anderson, R. L.; Dahms, A. S. Meth. Enzymol. 1975, 42C, 305.) The 2-keto acids 3-deoxy-D-glycero-pentulosonate and 3-deoxy-L-glycero-pentulosonate formed during enzyme-catalyzed dehydration are measured as semicarbazone derivatives. Two solutions are prepared and incubated separately at 30° C. for 3 min. The first solution (150 μL) contained Tris-HCl (50 mM), MgCl2 (10 mM) and an appropriate amount of cell lysate at pH 8.0. The second solution (25 μL) contained potassium D-xylonate or L-arabinonate (0.1 M). After the two solutions are mixed (time=0), aliquots (30 μL) are removed at timed intervals and mixed with semicarbazide reagent (200 μL), which contained 1% (w/v) of semicarbazide and 0.9% (w/v) of sodium acetate in water. Following incubation at 30° C. for 15 min, each sample is diluted to 1 mL with H2O. After removing the precipitated protein by centrifugation, the absorbance of semicarbazone is measured at 250 nm. One unit of D-xylonate or L-arabinonate dehydratase activity is defined as the formation of 1 μmol of 3-deoxy-D-glycero- or 3-deoxy-L-glycero-pentulosonate per min at 30° C. A molar extinction coefficient of 10,200 M−1cm−1 (250 nm) is used for 2-keto acid semicarbazone derivatives.

Provenance & Evidence