title: New insights on the mechanism of the K(+-) independent activity of crenarchaeota pyruvate kinases.
abstract: Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge-independent catalysis.
authors: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Distrito Federal, México.
authors: Gustavo
authors: G
authors: De la Vega-Ruíz
authors: Departamento de Ciencias Químico-Biológicas, Universidad de las Américas-Puebla, Ex-Hacienda Santa Catarina Mártir, Cholula, 72820 Puebla, México.
authors: Lenin
authors: L
authors: Domínguez-Ramírez
authors: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Distrito Federal, México.
authors: Héctor
authors: H
authors: Riveros-Rosas
authors: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Distrito Federal, México.
authors: Carlos
authors: C
authors: Guerrero-Mendiola
authors: Departamento de Bioquímica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 Distrito Federal, México.
authors: Alfredo
authors: A
authors: Torres-Larios
authors: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Distrito Federal, México.
authors: Gloria
authors: G
authors: Hernández-Alcántara
authors: Departamento de Biología, Facultad de Química, Universidad Nacional Autónoma de México, 04510 Distrito Federal, México.
authors: José J
authors: JJ
authors: García-Trejo
authors: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Distrito Federal, México.
authors: Leticia
authors: L
authors: Ramírez-Silva
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D019615:Crenarchaeota / Q000145:classification / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D010802:Phylogeny; D011188:Potassium / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D011770:Pyruvate Kinase; D011188:Potassium