Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate and adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate. New nucleotide affinity labels which react with rabbit muscle pyruvate kinase.
Three new reactive nucleotide analogues with bromo-keto substituents adjacent to a thiophosphate have been synthesized. Guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (GMPS-BDB), reacts covalently with rabbit muscle pyruvate kinase with complete inactivation and incorporation of 1.8 mol of reagent/mol of enzyme subunit. By contrast, the mono-keto compound, guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPS-BOP), causes no loss of pyruvate kinase activity. When the analogous adenosyl nucleotide derivatives are incubated with pyruvate kinase, the di-keto compound, adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)]thiophosphate (AMPS-BDB), rapidly effects inactivation, whereas the mono-keto compound, adenosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (AMPS-BOP), causes no loss of activity. Complete protection against inactivation by GMPS-BDB is provided by phosphoenolpyruvate in the presence of K+ and Mn2+ and the amount of reagent incorporated (0.9 mol/reagent/mol subunit) is reduced to half that observed in the absence of protectants. Gas-phase sequencing of the tryptic peptides purified from inactive GMPS-BDB or AMPS-BDB-modified enzyme gave the cysteine-labeled peptides: C151DENILWLDYK161, and N162IC164K165 as the two major peptide products, with a smaller amount of N43TGIIC48TIGPASR55. Reaction in the presence of the protectants PEP, K+, and Mn2+ yielded Cys164 as the only labeled residue, indicating that inactivation is primarily due to modification of Cys151. We propose that GMPS-BDB (or AMPS-BDB), which may exist in enolized form in aqueous solution, functions as a reactive analogue of phosphoenolpyruvate and GDP (ADP) to target Cys151 in the active site of pyruvate kinase.
Vollmer
S H
SH
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Walner
M B
MB
Tarbell
K V
KV
Colman
R F
RF
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels / Q000138:chemical synthesis*; D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006157:Guanosine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000138:chemical synthesis* / Q000378:metabolism
D000345:Affinity Labels; D007202:Indicators and Reagents; D010446:Peptide Fragments; D013873:Thionucleotides; C086304:adenosine 5'-O-(S-(4-bromo-2,3-dioxobutyl))thiophosphate; C086303:guanosine 5'-O-(S-(4-bromo-2,3-dioxobutyl))thiophosphate; D000244:Adenosine Diphosphate; D006157:Guanosine Monophosphate; D011770:Pyruvate Kinase