title: Pyruvate kinase revisited: the activating effect of K+.
abstract: 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.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México.
authors.forename: Jesús
authors.initials: J
authors.lastname: Oria-Hernández
authors.forename: Nallely
authors.initials: N
authors.lastname: Cabrera
authors.forename: Ruy
authors.initials: R
authors.lastname: Pérez-Montfort
authors.forename: Leticia
authors.initials: L
authors.lastname: Ramírez-Silva
mesh_terms: D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013463:Sulfuric Acid Esters; D014867:Water
chemical_list: D008024:Ligands; D013463:Sulfuric Acid Esters; D014867:Water; D011770:Pyruvate Kinase; D008274:Magnesium; C033522:methyl sulfate; D011188:Potassium