Pyruvate kinase revisited: the activating effect of K+. 
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.