Centro de Investigación Príncipe Felipe, Avenida Autopista del Saler 16, 46012 Valencia, Spain.
J Mol Biol. 2010 Dec 17;404(5):890-901. doi: 10.1016/j.jmb.2010.10.019. Epub 2010 Oct 21.
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 coli 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 β-sheet 4 and α-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. coli 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.
脯氨酸是细菌谷氨酸激酶（GK）和植物吡咯啉-5-羧酸合成酶（P5CS）的反馈抑制剂，是细胞渗透和氧化还原平衡的关键调节剂。使用动力学测定、定点突变、结构活性分析和对接计算，我们在三种三维结构中鉴定了该代谢物在大肠杆菌和空肠弯曲菌 GK 中的结合位点。脯氨酸结合腔部分与谷氨酸底物位点重叠，并且脯氨酸和谷氨酸与 GK 的相互作用受连接活性中心腔中β-片层 4 和α-螺旋 E 的 16 残基环调节。该环对于植物和人类 P5CS 的调节也很关键。此外，我们的结果表明，大肠杆菌酶的功能单元是二聚体，包含一个分子间氢键网络，将单体的活性中心腔相互连接，并且对底物结合很重要。
