Affinity labeling of rabbit muscle pyruvate kinase with dialdehyde-ADP.
Periodate-oxidized ADP (dialdehyde-ADP) inactivates rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and combines irreversibly to the enzyme. This inactivation is first-order with respect to dialdehyde-ADP and follows saturation kinetics, indicating that the enzyme first forms a reversible complex with the inactivator. Low Mg2+ concentrations stimulate the rate of inactivation, while higher concentrations have a protective effect. ADP and ATP, especially in the presence of Mg2+, protect very strongly against inactivation, while phosphoenolpyruvate and pyruvate are less effective. Dialdehyde-ADP is not a substrate, but acts as competitive inhibitor of ADP, with a KI of 4.5 mM. The analog has somewhat lower affinity to the enzyme than Mg-ADP, which has a Kd of 1.2 mM. Based on kinetic data, it is shown that one molecule of reagent must combine per enzyme active site in order to inactivate the enzyme. Incorporation of [14-C]dialdehyde-ADP to the enzyme and treatment of the data by the Tsou plot shows that 6-7 residues per subunit react with the modifier, two of them being essential for activity. From the evidence presented it is concluded: (1) dialdehyde-ADP behaves as an affinity label of rabbit muscle pyruvate kinase; (2) the inactivator binds probably to lysine residues at or near the active site, forming morpholine-like structures, and (3) the enzyme possesses two modifiable groups essential for activity, the reaction of one of them being sufficient to cause total loss in activity.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives*; D000345:Affinity Labels; D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors; D011817:Rabbits
D000345:Affinity Labels; D000244:Adenosine Diphosphate; C018808:adenosine 5'-diphosphate 2',3'-dialdehyde; D011770:Pyruvate Kinase