Serimbetov Zhalgas, Baxter Nicola J, Cliff Matthew J, Waltho Jonathan P
Manchester Institute of Biotechnology and School of Chemistry, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
Biomol NMR Assign. 2017 Oct;11(2):251-256. doi: 10.1007/s12104-017-9758-3. Epub 2017 Sep 2.
Human phosphoglycerate kinase (PGK) is an energy generating glycolytic enzyme that catalyses the transfer of a phosphoryl group from 1,3-bisphosphoglycerate (BPG) to ADP producing 3-phosphoglycerate (3PG) and ATP. PGK is composed of two α/β Rossmann-fold domains linked by a central α-helix and the active site is located in the cleft formed between the N-domain which binds BPG or 3PG, and the C-domain which binds the nucleotides ADP or ATP. Domain closure is required to bring the two substrates into close proximity for phosphoryl transfer to occur, however previous structural studies involving a range of native substrates and substrate analogues only yielded open or partly closed PGK complexes. X-ray crystallography using magnesium trifluoride (MgF) as a isoelectronic and near-isosteric mimic of the transferring phosphoryl group (PO), together with 3PG and ADP has been successful in trapping human PGK in a fully closed transition state analogue (TSA) complex. In this work we report the H, N and C backbone resonance assignments of human PGK in the solution conformation of the fully closed PGK:3PG:MgF:ADP TSA complex. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97% of all backbone resonances were assigned in the complex, with 385 out of a possible 399 residues assigned in the H-N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS-N webserver is in good agreement with the published X-ray crystal structure of this complex.
人磷酸甘油酸激酶（PGK）是一种产生能量的糖酵解酶，它催化将磷酰基从1,3 - 二磷酸甘油酸（BPG）转移到ADP上，生成3 - 磷酸甘油酸（3PG）和ATP。PGK由两个通过中央α - 螺旋连接的α/β罗斯曼折叠结构域组成，活性位点位于结合BPG或3PG的N结构域与结合核苷酸ADP或ATP的C结构域之间形成的裂隙中。需要结构域闭合以使两种底物紧密靠近从而发生磷酰基转移，然而先前涉及一系列天然底物和底物类似物的结构研究仅产生了开放或部分闭合的PGK复合物。使用三氟化镁（MgF）作为转移磷酰基（PO）的等电子和近等排模拟物，结合3PG和ADP的X射线晶体学已成功将人PGK捕获在完全闭合的过渡态类似物（TSA）复合物中。在这项工作中，我们报告了在完全闭合的PGK:3PG:MgF:ADP TSA复合物溶液构象中人PGK的H、N和C主链共振归属。归属通过异核多维核磁共振光谱获得。在该复合物中总共归属了所有主链共振的97%，在H - N TROSY谱中399个可能的残基中有385个被归属。使用TALOS - N网络服务器通过化学位移分析预测溶液二级结构与该复合物已发表的X射线晶体结构高度一致。
