Glucose-l-phosphate thymidylyltransferase

Glucose-1-phosphate thymidylyltransferase catalyzes the first of four steps in the biosynthesis of dTDP-L-rhamnose. The reaction is between dTTP and a-D-glucose 1-phosphate to form dTDP-D-glucose and PPj. 

Separation of dTTP, dTDP-D-glucose, dTDP, and dTMP (where dTMP, dTDP, and dTTP are the deoxy forms of thymidine mono-, di-, and triphosphate, respectively) occurred on a Supelcosil, LC-SAX column (4.6 mm x 250 mm). The chromatogram was developed at room temperature with a 20 mL linear gradient from 50 to 400 mM potassium phosphate buffer (pH 

at a flow rate of 1.5 mL/min. Quantitation was based on the absorbance at 254 nm, and all thymidine derivatives were monitored. 

The reaction mixture contained in a total volume of 300 /xL 15 /xmol Tris-HC1 buffer (pH 8.0), 3.6 /xmol of MgCfe, 7.2 fimol of a-D-glucose-l-phosphate, 1.8 /xmol of dTTP, 1.8 units of inorganic pyrophosphatase, and 30 /xL of appropriately diluted enzyme. The mixture was incubated at 37°C. Samples (36 /xL) were withdrawn at timed intervals up to 20 minutes, and the reaction was terminated by mixing with 1.0 mL of 50 mM potassium phosphate (pH 

Reactions deviated only slightly from linearity for up to 40% conversion of the starting amount of dTTP. 

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See also in sourсe #XX -- [ ]

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