Synthesis and characterization of diastereomers of guanosine 5'-O-(1-thiotriphosphate) and guanosine 5'-O-(2-thiotriphosphate).
The synthesis and characterization of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) using chemical and enzymatic methods are described. GTP alpha S A (SP diastereomer) can be prepared enzymatically from a chemically synthesized mixture of the diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S) with phosphoglycerate kinase. GTP alpha S B (RP diastereomer) can be similarly synthesized with succinyl-CoA synthetase and by back-digesting the small amounts of GTP alpha S A formed with phosphoglycerate kinase. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) serves as the precursor for both GTP beta S A (SP diastereomer), prepared with pyruvate kinase and by back-digesting with glycerol kinase, and GTP beta S B (RP diastereomer), obtained with acetate kinase and by back-digesting with myosin. These analogues can be gamma-32P labeled by 32Pi exchange with either phosphoglycerate kinase-phosphoglyceraldehyde dehydrogenase or succinyl-CoA synthetase. Finally, the interaction of these four nucleotides with acetate kinase, RNA polymerase, and succinyl-CoA synthetase is described.
D000084:Acetate Kinase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D066298:In Vitro Techniques; D007700:Kinetics; D008722:Methods; D013237:Stereoisomerism; D013379:Substrate Specificity; D013389:Succinate-CoA Ligases / Q000378:metabolism; D013873:Thionucleotides / Q000138:chemical synthesis*
D013873:Thionucleotides; C034882:guanosine 5'-O-(1-thiotriphosphate); C034883:guanosine 5'-O-(2-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000084:Acetate Kinase; D012321:DNA-Directed RNA Polymerases; D013389:Succinate-CoA Ligases