Molecular Mechanisms Modulating Glutamate Kinase Activity. Identification of the Proline Feedback Inhibitor Binding Site

Proline, the feedback inhibitor of bacterial glutamate kinase (GK) and plant pyrroline-5-carboxylate synthase (P5CS) enzymes, is a key regulator of the osmotic and redox balance of cells Using kinetic assays, site-directed mutagenesis, structure activity analyses, and docking calculations, we have identified the binding site of this metabolite in three-dimensional structures of Escherichia cob and Campylobacter jejuni GKs The proline-binding cavity partially overlaps with the glutamate substrate site, and the interaction of both proline and glutamate with GK is modulated by a flexible, 16-residue loop linking beta-sheet 4 and alpha-helix E in the active-center cavity This loop is also critical for regulation of plant and human P5CSs Furthermore, our results indicate that the functional unit of the E colt enzyme is dimeric and contains an intermolecular hydrogen-bond network that interconnects the active-center cavities of the monomers and is important for substrate binding (C) 2010 Elsevier Ltd All rights reserved