Reaction of pyruvate kinase with the new nucleotide affinity labels 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate.
Two new reactive nucleotides have been synthesized and characterized: 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate (8-BDB-TADP and 8-BDB-TATP). ADP or ATP was converted to 8-thio-ADP (-ATP) via 8-bromo-ADP (-ATP), followed by condensation with 1,4-dibromobutanedione. Rabbit muscle pyruvate kinase is inactivated by both reagents in a biphasic manner with an initial rapid loss of 75% activity, followed by a slow total inactivation. The initial fast reaction with both compounds exhibits nonlinear dependence on reagent concentration, indicating formation of a reversible enzyme-reagent complex prior to covalent attachment. The presence of the gamma-phosphoryl group improves the performance of the affinity label: KI values for the fast phase are similar (about 100 microM), whereas kmax for 8-BDB-TATP is about three times greater than that of 8-BDB-TADP (0.286 min-1 vs 0.0835 min-1). After an 80-min incubation with 175 microM of either reagent, about 2 mol/mol of subunit is incorporated with 76% inactivation caused by 8-BDB-TADP and 97% inactivation by 8-BDB-TATP. Loss of activity is prevented by substrates, with the best protection afforded by a combination of ATP, Mn2+, K+, and phosphoenolpyruvate. Reaction of pyruvate kinase with either compound in the presence of protecting ligands leads to incorporation of about 1 mol of reagent/mol of subunit with only about 15% loss of activity. These results suggest that 8-BDB-TADP and 8-BDB-TATP react with two groups on the enzyme, one of which is at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)
DeCamp
D L
DL
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Lim
S
S
Colman
R F
RF
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013873:Thionucleotides / Q000378:metabolism / Q000494:pharmacology
D000345:Affinity Labels; D008024:Ligands; D013873:Thionucleotides; C058329:8-((4-bromo-2,3-dioxobutyryl)thio)adenosine 5'diphosphate; C058333:8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase