title: The active site of pyrophosphate-dependent phosphofructo-1-kinase based on site-directed mutagenesis and molecular modeling.
abstract: Despite a low level of overall sequence identity between PPi-dependent and ATP-dependent phosphofructo-1-kinases (PFKs), similarities in active-site residues permit a convincing amino acid alignment of these two groups of kinases. Employing recent protein sequence and site-directed mutagenesis data along with the known three-dimensional coordinates of Escherichia coli ATP-dependent PFK, a model of the active site of PPi-dependent PFK was proposed. In addition to providing compatible placement of residues shown to be important by earlier mutagenesis studies, the model predicted an important role for two arginyl residues that are conserved in all known PPi-PFK sequences. Replacement by site-directed mutagenesis of these two residues with neutral amino acids in the PPi-PFK of Naegleria fowleri resulted in a substantial reduction in kcat while not altering the global structure of the enzyme. While the data indicate many similarities in the active-site structures and mechanisms of ATP-dependent and PPi-dependent PFKs, subtle differences, such as the relative roles of Arg residues in the active sites, have evolved in the development of these two subgroups of the PFK family.
authors.affiliation: Department of Biological Chemistry, Chicago Medical School, Illinois 60064, USA.
authors.forename: R M
authors.initials: RM
authors.lastname: Hinds
authors.forename: J
authors.initials: J
authors.lastname: Xu
authors.forename: D E
authors.initials: DE
authors.lastname: Walters
authors.forename: R G
authors.initials: RG
authors.lastname: Kemp
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism*; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; D010732:Phosphofructokinase-1; C068652:1-phosphofructokinase