title: 2-[(4-Bromo-2,3-dioxobutyl)thio]adenosine 5'-monophosphate, a new nucleotide analogue that acts as an affinity label of pyruvate kinase.
abstract: A new reactive adenine nucleotide has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-adenosine 5'-monophosphate (2-BDB-TAMP). Adenosine 5'-monophosphate 1-oxide was synthesized by reaction of AMP with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 5'-monophosphate (TAMP). The final product was generated by reaction of TAMP with 1,4-dibromobutanedione. The structure of 2-BDB-TAMP was determined by UV, 1H NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Rabbit muscle pyruvate kinase is inactivated by 2-BDB-TAMP at pH 7.0 and 25 degrees C. The inactivation rate exhibits a nonlinear dependence on the reagent concentration with KI = 0.57 mM. Protection against inactivation is provided by ADP and ATP, in the presence of Mn2+, as well as by phosphoenolpyruvate, in the presence of K+; in addition, partial protection is provided by AMP plus Mn2+. Incubation of pyruvate kinase with 0.075 mM 2-BDB-TAMP for 70 min in the absence of protective ligands leads to incorporation of 1.55 mol of reagent/mol of enzyme subunit when the enzyme is 53% inactive. In the presence of ADP and Mn2+, only 0.96 mol of reagent/mol of subunit is incorporated at 70 min, while the enzyme retains 100% activity. Similar results were obtained in the presence of ATP plus Mn2+. Assuming that the groups modified in the absence of ligands include those modified in the presence of the nucleotides, the 53% inactivation can be attributed to the modification of 0.59 (1.55-0.96) group per enzyme subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism; D000818:Animals; D066298:In Vitro Techniques; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013873:Thionucleotides
chemical_list: D000227:Adenine Nucleotides; D000345:Affinity Labels; D013873:Thionucleotides; D000249:Adenosine Monophosphate; D008345:Manganese; C048059:2-((4-bromo-2,3-dioxobutyl)thio)adenosine 5'-monophosphate; D011770:Pyruvate Kinase