Stereochemical Courses of Nucleotidyltransferase and Phosphotransferase Action. Uridine Diphosphate Glucose Pyrophosphorylase, Galactose-1-phosphate Uridylyltransferase, Adenylate Kinase, and Nucleoside Diphosphate Kinase†

Reactions catalyzed by UDP-glucose pyrophosphorylase, galactose-1-P uridylyltransferase, and nucleoside diphosphate kinase can be analyzed for their stereochemical courses, by using nuclear magnetic resonance and mass spectroscopic techniques with either sulfur or sulfur and 18O-substituted nucleotides as substrates. The UDP-glucose pyrophosphorylase reaction proceeds with inversion of configuration at Pa of uridine-5'-(l-thiodiphospho)glucose, and the galactose-1-P uridylyltransferase reaction proceeds with retention. Therefore, the bond cleavage/formation mechanisms for these chemically matched reactions cannot be identical. The simplest explanation for the stereochemical results is that the former reaction involves a single displacement at Pα and the latter involves a double-displacement mechanism. Thiophosphoryl group transfer by nucleoside diphosphate kinase acting on the [18O]phosphorothioate substrate analogue of ATP chirally substituted at Pγ proceeds with retention of configuration, in contrast to the inversion of configuration observed earlier in the adenylate kinase reaction [Richard, J. P., & Frey, P. A. (1978) J. Am. Chem. Soc. 100, 7757], As in the uridylyltransferases, these chemically matched reactions occur by different mechanisms, the former by a double and the latter by a single displacement at Pγ of ATP. UDP-glucose pyrophosphorylase accepts uridine 5'-(l-thiophosphate) having the R configuration at Pa and uridine 5'-(l-thiodiphospho)-glucose with the S configuration at Pα as substrates, while galactose-1-P uridylyltransferase accepts only the R configuration at Pa of uridine 5'-(l-thiodiphospho)glucose or -galactose. By use of these stereochemical preferences Pα epimers of uridine 5'-(l-thiotriphosphate) and uridine 5'-(l-thiodi-phosphate)glucose can be prepared in pure form from synthetic mixtures.