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Perez Montfort, R; Oria-Hernandez, J; Cabrera, N; Ramirez-Silva, L (2005)


J BIOL CHEM 280(45):37924-37929
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For more than 50 years, it has been known that K+ is an essential activator of pyruvate kinase (Kachmar, J. F., and Boyer, P. D. ( 1953) J. Biol. Chem. 200, 669 - 683). However, the role of K+ in the catalysis by pyruvate kinase has not been totally understood. Previous studies without K+ showed that the affinity of ADP-Mg2+ depends on the concentration of phosphoenolpyruvate, although the kinetics of the enzyme at saturating K+ concentrations show independence in the binding of substrates (Reynard, A. M., Hass, L. F., Jacobsen, D. D. & Boyer, P. D. ( 1961) J. Biol. Chem. 236, 2277 - 2283). Here, we explored the kinetics of the enzyme with and without K+. The results show that without K+, the kinetic mechanism of pyruvate kinase changes from random to ordered with phosphoenolpyruvate as first substrate. V-max with K+ was about 400 higher than without K+. In the presence of K+, the affinities for phosphoenolpyruvate, ADP-Mg2+, oxalate, and ADP-Cr2+ were 2 - 6-fold higher than in the absence of K+. This as well as fluorescence data also indicate that K+ is involved in the acquisition of the active conformation of the enzyme, allowing either phosphoenolpyruvate or ADP to bind independently ( random mechanism). In the absence of K+, ADP cannot bind to the enzyme until phosphoenolpyruvate forms a competent active site ( ordered mechanism). We propose that K+ induces the closure of the active site and the arrangement of the residues involved in the binding of the nucleotide.