Affinity labeling of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase with the 2',3'-dialdehyde derivative of ATP.
Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] is completely inactivated by the 2',3'-dialdehyde derivative of ATP (oATP) in the presence of Mn2+. The dependence of the pseudo-first-order rate constant on reagent concentration indicates the formation of a reversible complex with the enzyme (Kd = 60 +/- 17 microM) prior to covalent modification. The maximum inactivation rate constant at pH 7.5 and 30 degrees C is 0.200 +/- 0.045 min-1. ATP or ADP plus phosphoenolpyruvate effectively protect the enzyme against inactivation. oATP is a competitive inhibitor toward ADP, suggesting that oATP interacts with the enzyme at the substrate binding site. The partially inactivated enzyme shows an unaltered Km but a decreased V as compared with native phosphoenolpyruvate carboxykinase. Analysis of the inactivation rate at different H+ concentrations allowed estimation of a pKa of 8.1 for the reactive amino acid residue in the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of about one mole of [8-14C]oATP per mole of enzyme subunit. The results indicate that oATP can be used as an affinity label for yeast phosphoenolpyruvate carboxykinase.
Saavedra
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C
Departamento de Química, Facultad de Ciencia, Universidad de Santiago de Chile.
Araneda
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S
Cardemil
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E
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D001665:Binding Sites / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000345:Affinity Labels; C017199:2',3'-dialdehyde ATP; D000255:Adenosine Triphosphate; D010729:Phosphoenolpyruvate Carboxykinase (GTP)