Crystallographic Studies of Phosphoryl Group Transfer Reactions

参与磷转移反应的蛋白质的三维结构将由x射线晶体学确定，作用机制将被研究，补充晶体学工作与定点诱变和动力学表征。本研究的重点是三种酶：(1)丙酮酸磷酸二激酶（PPDK），它通过三磷酸基转移反应途径催化ATP和PEP两种高能化合物的相互转化。来自共生梭菌的细菌酶的结构已经在实验室中被确定（Herzberg等人，PNAS 93:2652, 1996），揭示了一个三结构域分子，其中丙酮酸和核苷酸活性位点位于45个奥格斯特罗姆单位之间。该结构的含义是，磷酸化基团从一个底物转移到另一个底物时，伴随着两个远端活性位点之间的结构域旋转，这一机制为其他复杂的多结构域蛋白的通信提供了一个有吸引力的范式。我们将进一步研究底物和抑制剂与PPDK的结合及其巨大的构象转变，利用细菌蛋白催化ATP形成方向的反应，以及迷宫PPDK在生理条件下催化PEP形成的逆反应，并通过苏氨酸残基上的特定磷酸化来调节。(2)磷酸烯醇丙酮酸磷酸化酶，催化PEP重排为磷酸丙酮酸。该反应是合成天然膦酸盐的主要入口步骤，这种化合物具有广泛的生物活性。了解该酶的催化机制对开发合成和分解目标膦酸盐的方法具有重要意义。测定了咸水贻贝PEP突变酶的晶体结构。活性位点结构的详细视图将有助于阐明催化机制，并将为位点定向诱变实验和晶体结合研究提供结构基础，旨在证实或反驳将推断的机制。(3)磷酸烯醇丙酮酸糖磷酸转移酶系统（PTS）是一种涉及多种磷酸转移反应的细菌多酶糖转运系统。在之前的资助期内，系统的两个蛋白质成分HPr和葡萄糖渗透酶的IIA结构域的结构确定之后，重点将转移到酶I上，它从PEP中接受磷酸化基团并将其传递给HPr。一个截断版本的酶I已经结晶，它包含pep结合域和磷酸化基团受体域。结构测定将揭示与抑制剂mg -草酸盐的结合模式，以及与底物PEP的相互作用。这项研究的目的是为了更好地理解蛋白质磷酸化调控的本质，这是一个广泛的生物过程的中心反应。结合x射线晶体学和蛋白质工程技术来阐明蛋白质的三维结构，并通过晶体中的结合研究和定点诱变方法来探测它们的功能。本文选择了三种利用磷酸转移反应的系统进行研究：(a)在一些细菌和植物中催化ATP和PEP两种高能化合物相互转化的丙酮酸磷酸二激酶。(b) PEP变化酶，在合成膦酸盐、具有抗生素、抗真菌、杀虫、抗病毒、降压、除草和神经毒性的化合物中催化第一反应。(c)在细菌中介导糖跨膜摄取的磷酸化转移链。链上的第一个蛋白质，酶I，从高能化合物磷酸烯醇丙酮酸中接受一个磷酸基，并将其传递给后面的蛋白质HPr，将被研究。

9813271 Osnat Herzberg The three dimensional structures of proteins involved in phosphotransfer reactions will be determined by X-ray crystallography and the mechanism of action will be investigated, complementing the crystallographic work with site-directed mutagenesis and kinetic characterization. The focus of this study is on three enzymes: (1) Pyruvate phosphate dikinase (PPDK) which catalyzes the inter-conversion of the two high-energy compounds, ATP and PEP, via a three phosphoryl transfer reaction pathway. The structure of the bacterial enzyme from Clostridium symbiosum has been determined in the laboratory (Herzberg et al., PNAS 93:2652, 1996), revealing a three-domain molecule in which the pyruvate and nucleotide active sites are located 45 Augstrom units apart. The implication of the structure is that phosphoryl group transfer from one substrate to the other are accompanied by a domain swiveling between the two remote active sites, a mechanism that offers an attractive paradigm for communication in other complex multidomain proteins. The substrate and inhibitor binding to PPDK and the dramatic conformational transition will be further investigated using the bacterial protein that catalyzes the reaction in the direction of ATP formation, and the maze PPDK which catalyzes the reverse reaction toward PEP formation under physiological conditions, and which is regulated by a specific phosphorylation on a threonine residue. (2) Phosphoenolpyruvate phosphomutase which catalyses the rearrangement of PEP to phosphonopyruvate. This reaction serves as the major entry step into the synthesis of natural phosphonates, compounds that display a wide spectrum of biological activities. Understanding of the catalytic mechanism of the enzyme is important for the development of methods for synthesizing and breaking down target phosphonates. The crystal structure of PEP mutase from salt-water mussel will be determined. A detailed view of the architecture of the active site will help in the elucidation of the catalytic mechanism, and will provide the structural basis for site-directed mutagenesis experiments and crystallographic binding studies designed to confirm or refute the mechanism that will be deduced. (3) The phosphoenolpyruvate: sugar phosphotransferase system (PTS) which is a bacterial multi-enzyme sugar transport system that involves multi-phosphotransfer reactions. Following the structure determination of two protein components of the system during previous funding periods, HPr and the IIA domain of the glucose permease, the focus will be shifted to Enzyme I which accepts a phosphoryl group from PEP and delivers it to HPr. A truncated version of enzyme I has been crystallized, which contains the PEP-binding domain and the phosphoryl group acceptor domain. The structure determination will reveal the mode of binding with an inhibitor, Mg-oxalate, and by analogy, the interaction with the substrate, PEP. The goal of this study is to gain better understanding of the nature of regulation by protein phosphorylations, reactions which are central to a wide range of biological processes. A combination of X-ray crystallography and protein engineering techniques is used to elucidate the three dimensional structure of the proteins, and to probe their function by binding studies in the crystals, and by site-directed mutagenesis approaches. Three systems that utilize phosphotransfer reactions are selected for study: (a) The enzyme pyruvate phosphate dikinase that catalyses the inter-conversion of two high energy compounds, ATP and PEP, in some bacteria and plants. (b) The enzyme PEP mutase that catalyzes the first reaction in the synthesis of phosphonates, compounds with antibiotic, antifungal, insecticidal, antiviral, antihypertensive, herbicidal, and neurotoxic functions. (c) A phosphoryl transfer chain that mediates sugar uptake across the membrane in bacteria. The first protein in the chain, Enzyme I, which accepts a phosphoryl group from the high-energy compound phosphoenolpyru vate and delivers it to the proceeding protein, HPr, will be investigated.