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Mapping active site residues in glutamate‐5‐kinase. The substrate glutamate and the feed‐back inhibitor proline bind at overlapping sites
Author(s) -
Pérez-Arellano Isabel,
Rubio Vicente,
Cervera Javier
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.10.031
Subject(s) - proline , active site , biochemistry , binding site , glutamate receptor , substrate (aquarium) , enzyme , chemistry , transferase , biology , amino acid , stereochemistry , receptor , ecology
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 ∼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.