TDP-Glucose

The reactions were carried out under standard conditions using the specifically tritiated substrate mixed with uniformly 14C labeled TDP-glucose. Samples were taken at different time intervals and the reaction products were isolated. 14C activity was used to measure the reaction coordinate. The reactions were permitted to proceed until isotopic equilibrium was achieved. 

Several independent lines of experimental evidence have been given (41, 42) to substantiate the proposed reaction mechanism for UDP-galactose-4-epimerase, shown in Figure 9. Recently, Kalckar and coworkers (42) used TDP-glucose-4T as a substrate. The initial attack occurs at carbon 4 and results in conversion to the enzyme-bound-4-ulose intermediate, accompanied by formation of enzyme-NADT. The 4-ulose intermediate serves as hydrogen acceptor to restore enzyme-NAD+ and to release product. 

After E. coli pyrophosphatase and 32Pj were mixed with the appropriate substrates and purified enzymes at pH 8.5, reduction of TPN+ proceeded with eventual trapping of 10% of the 3ZPi as thymidine triphosphate (TP3ZP3ZP). The net stoichiometry of the coupled reactions was 3ZP, —0.51 /xmole TP3ZP3ZP, +0.27 /xmole TDP-glucose, —0.26 /xmole and TPNH, +0.21 /xmole 12). 

The product of the thymidylyltransferase reaction, TDP-glucose, is a natural substrate for a well-known reaction catalyied by TDP-glucose 4,6-dehydratase (306). The TDP-glucose 4,6-dehydratases from both. StreptfTmyces sp.. srrain C5 and. S. peurediu, purified to homogeneity, were homodimers with subunit values of of 39,OCX) and 36,000,... 

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