title: Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis.
abstract: The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP(-) and was activated by free Mg(2+). On the other hand, HXE mutants did not affect the KM for MgADP(-), were still inhibited by free Mg(2+), and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP-AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago 7800003, Chile.
authors.forename: María José
authors.initials: MJ
authors.lastname: Abarca-Lagunas
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago 7800003, Chile.
authors.forename: Jaime Andrés
authors.initials: JA
authors.lastname: Rivas-Pardo
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago 7800003, Chile. Electronic address: ceramirez@uchile.cl.
authors.forename: César A
authors.initials: CA
authors.lastname: Ramírez-Sarmiento
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago 7800003, Chile. Electronic address: vguixe@uchile.cl.
authors.forename: Victoria
authors.initials: V
authors.lastname: Guixé
mesh_terms: D020816:Amino Acid Motifs; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D017124:Conserved Sequence; D005941:Glucokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D019843:Archaeal Proteins; C420842:ADP-dependent glucokinase; D005941:Glucokinase; D005947:Glucose
keywords: ADP-dependent kinase; Archaeal enzyme; Divalent metal cation; Glucokinase; Protein–ligand binding