NADPH → HCID 178434963, compound

Participants

Reaction Steps

A coupled assay was employed to assay YEF B activity. This was required since its substrate, GDP-4-keto-6-deoxymannose, is unstable and not commercially available. Twenty mUnit of GDP-mannose dehydratase was allowed to react with 480 nMole of GDP-mannose for 20 minutes under standard assay conditions as previously. At this time greater than 90% of the GDP-mannose has been converted to GDP-4-keto-6-deoxymannose, the substrate for YEF B. The following additions were then made: 500 nmole glucose, MgCl2 to 20 mM, NADPH to 0.15 mM, 10 mUnit glucose dehydrogenase, 100 nmole CTP (to normalize for losses that may occur when sample is injected onto HPLC), YEF B and water to 50 μl. The reaction was allowed to proceed for ten minutes at which point the reaction was stopped by freezing on dry ice. After thawing 10 μl aliquots were analyzed by reverse phase HPLC analysis. The amount of GDP fucose formed was calculated by analyzing a separate assay mix in the absence of GDP-mannose dehydratase to yield a 100% value for the absorption peak of GDP-mannose. This peak area was then divided by the peak area of GDP-fucose formed to yield percentage of GDP-fucose formed. (% GDP-fucose (nMole GDP-mannose in reaction)=nMole GDP-fucose formed.

Provenance & Evidence