title: Mapping active site residues in glutamate-5-kinase. The substrate glutamate and the feed-back inhibitor proline bind at overlapping sites.
abstract: Glutamate-5-kinase (G5K) catalyzes the controlling first step of proline biosynthesis. Substrate binding, catalysis and feed-back inhibition by proline are functions of the N-terminal approximately 260-residue domain of G5K. We study here the impact on these functions of 14 site-directed mutations affecting 9 residues of Escherichia coli G5K, chosen on the basis of the structure of the bisubstrate complex of the homologous enzyme acetylglutamate kinase (NAGK). The results support the predicted roles of K10, K217 and T169 in catalysis and ATP binding and of D150 in orienting the catalytic lysines. They support the implication of D148 and D150 in glutamate binding and of D148 and N149 in proline binding. Proline increases the S(0.5) for glutamate and appears to bind at a site overlapping with the site for glutamate. We conclude that G5K and NAGK closely resemble each other concerning substrate binding and catalysis, but that they have different mechanisms of feed-back control.
authors.affiliation: Centro de Investigación Príncipe Felipe, Avda. Autopista del Saler 16, Valencia 46013, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Pérez-Arellano
authors.forename: Vicente
authors.initials: V
authors.lastname: Rubio
authors.forename: Javier
authors.initials: J
authors.lastname: Cervera
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids; D001665:Binding Sites; D002384:Catalysis; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D025461:Feedback, Physiological; D018698:Glutamic Acid / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D016415:Sequence Alignment
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D011392:Proline; D017851:Phosphotransferases (Carboxyl Group Acceptor); C020650:glutamate 5-kinase; C018721:acetylglutamate kinase