Modification of human erythrocyte pyruvate kinase by an active site-directed reagent: bromopyruvate.
Human erythrocyte pyruvate kinase was modified with bromopyruvate and the kinetic behavior of the modified enzyme was investigated. When the enzyme was modified with bromopyruvate in the absence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate the inactivation followed a pseudo first-order kinetics. The inactivation rate constant, ks, was 1.84 +/- 0.15 min(-1). Kd of the bromopyruvate-enzyme complex was 0.14 +/- 0.03 mM. The presence of adenosine-5'-diphosphate, phosphoenolpyruvate or fructose-1,6-diphosphate in the modification medium or the presence of fructose-1,6-diphosphate in the assay medium resulted in deviation of the inactivation kinetics from pseudo first-order. Phosphoenolpyruvate was better than adenosine-5'-diphosphate for protection against bromopyruvate modification whereas fructose-1,6-diphosphate was ineffective. The modified enzyme showed negative cooperativity in the presence of fructose-1,6-diphosphate whereas in the absence of it no activity was detected.
Acan
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NL
Department of Biochemistry, Faculty of Medicine, Hacettepe University, Ankara, Turkey.
Ozer
N
N
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000746:Anemia, Hemolytic, Congenital Nonspherocytic / Q000201:enzymology; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D005635:Fructosediphosphates / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011773:Pyruvates / Q000494:pharmacology; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D005635:Fructosediphosphates; D011773:Pyruvates; D000244:Adenosine Diphosphate; C017092:bromopyruvate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate