| 16147999 |
Oria-Hernandez J, Cabrera N, Perez-Montfort R, Ramirez-Silva L: Pyruvate kinase revisited: the activating effect of K+. J Biol Chem. 2005 Nov 11;280(45):37924-9. Epub 2005 Sep 7.
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-Mg (2+) 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 phosphoenol-pyruvate as first substrate. V (max) with K (+) was about 400 higher than without K (+). In the presence of K (+), the affinities for phosphoenol-pyruvate, ADP-Mg (2+), oxalate, and ADP-Cr (2+) 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. |
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