Auerbach G, Huber R, Grättinger M, Zaiss K, Schurig H, Jaenicke R, Jacob U
Max-Planck-Institut für Biochemie, Abt. Strukturforschung, 82152, Martinsried, Germany.
Structure. 1997 Nov 15;5(11):1475-83. doi: 10.1016/s0969-2126(97)00297-9.
Phosphoglycerate kinase (PGK) is essential in most living cells both for ATP generation in the glycolytic pathway of aerobes and for fermentation in anaerobes. In addition, in many plants the enzyme is involved in carbon fixation. Like other kinases, PGK folds into two distinct domains, which undergo a large hinge-bending motion upon catalysis. The monomeric 45 kDa enzyme catalyzes the transfer of the C1-phosphoryl group from 1, 3-bisphosphoglycerate to ADP to form 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP. For decades, the conformation of the enzyme during catalysis has been enigmatic. The crystal structure of PGK from the hyperthermophilic organism Thermotoga maritima (TmPGK) represents the first structure of an extremely thermostable PGK. It adds to a series of four known crystal structures of PGKs from mesophilic via moderately thermophilic to a hyperthermophilic organism, allowing a detailed analysis of possible structural determinants of thermostability.
The crystal structure of TmPGK was determined to 2.0 A resolution, as a ternary complex with the product 3-phosphoglycerate and the product analogue AMP-PNP (adenylyl-imido diphosphate). The complex crystallizes in a closed conformation with a drastically reduced inter-domain angle and a distance between the two bound ligands of 4.4 A, presumably representing the active conformation of the enzyme. The structure provides new details of the catalytic mechanism. An inter-domain salt bridge between residues Arg62 and Asp200 forms a strap to hold the two domains in the closed state. We identify Lys197 as a residue involved in stabilization of the transition state phosphoryl group, and so term it the 'phosphoryl gripper'.
The hinge-bending motion of the two domains upon closure of the structure, as seen in the Trypanosoma PGK structure, is confirmed. This closed conformation obviously occurs after binding of both substrates and is locked by the Arg62-Asp200 salt bridge. Re-orientations in the conserved active-site loop region around Thr374 also bring both domains into direct contact in the core region of the former inter-domain cleft, to form the complete catalytic site. Comparison of extremely thermostable TmPGK with less thermostable homologues reveals that its increased rigidity is achieved by a raised number of intramolecular interactions, such as an increased number of ion pairs and additional stabilization of alpha helix and loop regions. The covalent fusion with triosephosphate isomerase might represent an additional stabilization strategy.
磷酸甘油酸激酶（PGK）在大多数活细胞中至关重要，它在需氧生物的糖酵解途径中参与ATP的生成，在厌氧生物中参与发酵。此外，在许多植物中，该酶还参与碳固定。与其他激酶一样，PGK折叠成两个不同的结构域，在催化过程中会发生大幅度的铰链弯曲运动。这种单体的45 kDa酶催化将1,3 - 二磷酸甘油酸的C1 - 磷酰基转移到ADP上，形成3 - 磷酸甘油酸和ATP。几十年来，该酶在催化过程中的构象一直是个谜。嗜热栖热菌（TmPGK）的PGK晶体结构代表了首个极其耐热的PGK结构。它补充了从嗜温、中度嗜温到嗜热生物的一系列四个已知PGK晶体结构，从而能够详细分析耐热性可能的结构决定因素。
TmPGK的晶体结构被确定为分辨率为2.0 Å的三元复合物，包含产物3 - 磷酸甘油酸和产物类似物AMP - PNP（腺苷酰亚胺二磷酸）。该复合物以封闭构象结晶，结构域间角度大幅减小，两个结合配体之间的距离为4.4 Å，推测代表了酶的活性构象。该结构提供了催化机制的新细节。残基Arg62和Asp200之间的结构域间盐桥形成一条带，将两个结构域保持在封闭状态。我们确定Lys197是参与稳定过渡态磷酰基的残基，因此将其称为“磷酰基抓手”。
正如在锥虫PGK结构中所见，证实了结构闭合时两个结构域的铰链弯曲运动。这种封闭构象显然在两种底物结合后出现，并由Arg62 - Asp200盐桥锁定。围绕Thr374的保守活性位点环区域的重新定向也使两个结构域在前结构域间裂隙的核心区域直接接触，形成完整的催化位点。将极其耐热的TmPGK与耐热性较低的同源物进行比较发现，其增加的刚性是通过增加分子内相互作用实现的，例如增加离子对的数量以及α螺旋和环区域的额外稳定。与磷酸丙糖异构酶的共价融合可能代表了一种额外的稳定策略。
