Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid.
Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.
D000478:Alkylation; D000645:Ammonium Sulfate; D000818:Animals; D001665:Binding Sites; D007651:Keto Acids / Q000138:chemical synthesis; D007700:Kinetics; D007982:Levulinic Acids; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D010421:Pentanoic Acids / Q000138:chemical synthesis; D011770:Pyruvate Kinase / Q000302:isolation & purification; D011773:Pyruvates; D014316:Tritium; D014631:Valerates / Q000138:chemical synthesis
D007651:Keto Acids; D007982:Levulinic Acids; D008024:Ligands; D010421:Pentanoic Acids; D011773:Pyruvates; D014631:Valerates; D014316:Tritium; C013439:5-chloro-4-oxopentanoic acid; D011770:Pyruvate Kinase; D000645:Ammonium Sulfate