title: Catalytic and regulatory roles of divalent metal cations on the phosphoryl-transfer mechanism of ADP-dependent sugar kinases from hyperthermophilic archaea.
abstract: In some archaea, glucose degradation proceeds through a modified version of the Embden-Meyerhof pathway where glucose and fructose-6-P phosphorylation is carried out by kinases that use ADP as the phosphoryl donor. Unlike their ATP-dependent counterparts these enzymes have been reported as non-regulated. Based on the three dimensional structure determination of several ADP-dependent kinases they can be classified as members of the ribokinase superfamily. In this work, we have studied the role of divalent metal cations on the catalysis and regulation of ADP-dependent glucokinases and phosphofructokinase from hyperthermophilic archaea by means of initial velocity assays as well as molecular dynamics simulations. The results show that a divalent cation is strictly necessary for the activity of these enzymes and they strongly suggest that the true substrate is the metal-nucleotide complex. Also, these enzymes are promiscuous in relation to their metal usage where the only considerations for metal assisted catalysis seem to be related to the ionic radii and coordination geometry of the cations. Molecular dynamics simulations strongly suggest that this metal is bound to the highly conserved NXXE motif, which constitutes one of the signatures of the ribokinase superfamily. Although free ADP cannot act as a phosphoryl donor it still can bind to these enzymes with a reduced affinity, stressing the importance of the metal in the proper binding of the nucleotide at the active site. Also, data show that the binding of a second metal to these enzymes produces a complex with a reduced catalytic constant. On the basis of these findings and considering evolutionary information for the ribokinase superfamily, we propose that the regulatory metal acts by modulating the energy difference between the protein-substrates complex and the reaction transition state, which could constitute a general mechanism for the metal regulation of the enzymes that belong this superfamily.
authors: Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile.
authors: Felipe
authors: F
authors: Merino
authors: Jaime Andrés
authors: JA
authors: Rivas-Pardo
authors: Andrés
authors: A
authors: Caniuguir
authors: Ivonne
authors: I
authors: García
authors: Victoria
authors: V
authors: Guixé
mesh_terms: D025181:ATP Synthetase Complexes / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D002413:Cations, Divalent / Q000378:metabolism; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D025221:Phosphofructokinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D002413:Cations, Divalent; D008670:Metals; D009711:Nucleotides; D011994:Recombinant Proteins; D025221:Phosphofructokinases; D005941:Glucokinase; D025181:ATP Synthetase Complexes