β-phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases

The α- and β-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyl-transferase reactions. The α analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the βS analogs were approximately 10 times more resistant. The βS analog and native UDP-Glc were found to have comparable K_m's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent V_max of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4β-galactosyltransferase exhibited a V_max with (βS) UDP-Gal that was only slightly lower than that with UDP-Gal and a K_m that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4β-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the βS analog in each system.