title: Synthesis and utilization of a nonhydrolyzable phosphoadenosine phosphosulfate analog.
abstract: 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) functions as the high-energy sulfate donor for sulfate ester synthesis in all higher organisms. This activated sulfate, like its adenosine 5'-phosphosulfate precursor, is both chemically labile and vulnerable to sulfohydrolase degradation. These obstacles have limited the utility of the native PAPS in the purification and mechanistic description of the numerous PAPS-utilizing enzymes. This paper describes the synthesis of the 2'- and 3'-isomers of a nonhydrolysable, and thus stable, PAPS analog, beta-methylene-PAPS, from the previously described beta-methylene-APS (L. Callahan et al., Anal. Biochem. 177, 67-71, 1989). The method involves phosphorylation of beta-methylene-APS with trimetaphosphate and separation of the resulting mixed 2'(3')-isomers by ion-pair reverse-phase HPLC. The utilization of this analog as an inhibitor of APS kinase and PAPS translocase, two of the numerous PAPS-utilizing activities, as well as an affinity ligand for purification of APS kinase, is described.
authors.affiliation: Department of Pediatrics, Joseph P. Kennedy Jr. Mental Retardation Research Center, University of Chicago, Illinois 60637.
authors.forename: K
authors.initials: K
authors.lastname: Ng
authors.forename: M
authors.initials: M
authors.lastname: D'Souza
authors.forename: L
authors.initials: L
authors.lastname: Callahan
authors.forename: D H
authors.initials: DH
authors.lastname: Geller
authors.forename: A E
authors.initials: AE
authors.lastname: Kearns
authors.forename: S
authors.initials: S
authors.lastname: Lyle
authors.forename: N B
authors.initials: NB
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000737:chemistry; D010724:Phosphoadenosine Phosphosulfate / Q000031:analogs & derivatives* / Q000378:metabolism; D010770:Phosphotransferases / Q000737:chemistry / Q000302:isolation & purification*; D017853:Phosphotransferases (Alcohol Group Acceptor)
chemical_list: D002352:Carrier Proteins; D010710:Phosphates; D010724:Phosphoadenosine Phosphosulfate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase