Crystallographic Studies of Phosphoryl Group Transfer Reactions

涉及磷酰基转移反应的蛋白质的三维结构将通过X射线晶体学测定，反应机理将使用晶体学和定点诱变方法的组合进行研究。 在上一个资助期间研究的磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）将进一步研究。 该项目还将包括另一种酶，丙酮酸磷酸二激酶（PPDK），其显示出与PTS的酶I的序列同源性。 PTS是一种多酶系统，通过磷酰基转移链调节糖跨膜转运。 在过去的两年中，我们已经确定了两个PTS组分，HPr和葡萄糖通透酶的IIA结构域的晶体结构，并且在原子水平上了解了它们之间的磷酰基转移反应的性质。 拟议的工作将借鉴这些发现，并测试我们的理解工程改变磷酸化特异性的葡萄糖IIA结构域。 设计的突变体将进行生化分析，以及通过X射线晶体学，以了解成功或失败的原因。 PTS研究的第二部分是通过MIR方法对磷酸转移链中的第一个蛋白质酶I进行结构测定，酶I最近已结晶。 这将提供一个完整的结构基础，为今后的研究系统的可溶性蛋白质。 结构测定之后将进行抑制剂结合研究，以阐明磷酸烯醇丙酮酸（PEP）磷酸化和磷酰基转移至HPr的机制。 第三部分完成了HPr高盐型和低盐型的结构比较，并对HPr突变体H15A进行了结构优化。 这项工作是重要的，以解决目前的争议有关的问题，是否磷酸化HPr是与骨架的构象转变。 我们的数据表明，情况并非如此。 除了关于临时秘书处的工作外，该项目的范围还扩大到包括PPDK，这是一种催化PEP和ATP相互转化的多域酶。 其与酶I的序列同源性源于共同底物PEP。 PPDK催化总共三个磷酰基转移，其中一个涉及焦磷酸中间体，这是酶促磷酰基转移反应中前所未有的中间体。 共生梭菌酶的结构测定将有助于抑制剂结合研究和诱变工作，探测这种复杂酶的各种机制方面。 与磷酰基转移，这是提出了大量的构象转变的性质，将揭示这些结构的研究。 拟议研究的目标是更好地了解蛋白质磷酸化调节的性质，这些反应是广泛的生物过程的核心。 X射线晶体学和蛋白质工程技术的组合将用于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。 已经选择了两种利用多磷酸转移反应的系统：（a）介导细菌跨膜糖摄取的磷酸转移链。 将研究涉及的三种可溶性蛋白质，酶I、HPr和酶IIA。(b)丙酮酸磷酸二激酶在某些细菌和植物中催化两种高能化合物ATP和PEP相互转化的酶。 ***

9316934 Herzberg The three dimensional structures of proteins involved in phosphoryl group transfer reactions will be determined by X-ray crystallography and the reaction mechanism will be investigated using a combination of crystallographic and site-directed mutagenesis approaches. The phosphoenolpyruvate:sugar phosphotransferase system (PTS) studied during the previous funding period will be investigated further. The project will also include another enzyme, pyruvate phosphate dikinase (PPDK), which shows sequence homology to Enzyme I of the PTS. The PTS is a multi- enzyme system that regulates sugar transport across the membrane by a phosphoryl group transfer chain. The crystal structures of two of the PTS components, HPr and the IIA domain of the glucose permease, have been determined by us during the last two years, and much learned about the nature of the phosphoryl transfer reaction between them at the atomic level. The proposed work will draw on these findings and test our understanding by engineering altered phosphorylation specificity of the glucose IIA domain. The designed mutants will be analyzed biochemically, as well as by X- ray crystallography, to understand the reasons for success or failure. The second part of the studies of the PTS is the structure determination by MIR methods of the first protein in the phosphotransfer chain, Enzyme I, which has been recently crystallized. This will provide a complete structural basis for future investigations of the soluble proteins of the system. The structure determination will be followed by inhibitor binding studies to elucidate the mechanism of phosphorylation by phosphoenolpyruvate (PEP) and of phosphoryl transfer to HPr. The third part is the completion of the structural comparison between the high and low salt forms of HPr, and the structure refinement of the HPr mutant H15A. This work is important for resolving a current controversy related to the question whether phosphorylation of HPr i s associated with a conformational transition of the backbone. Our data shows that this is not the case. In addition to work on the PTS, the scope of the project has been expanded to include PPDK, a multi-domain enzyme that catalyses the inter- conversion of PEP and ATP. Its sequence homology to Enzyme I stems from the common substrate, PEP. PPDK catalyses a total of three phosphoryl group transfers, one of which involves a pyrophosphate intermediate, an unprecedented intermediate in enzymatic phosphoryl transfer reactions. The structure determination of the Clostridium symbiosum enzyme will facilitate inhibitor binding studies and mutagenesis work that probe the various mechanistic aspects of this complex enzyme. The nature of the conformational transition associated with the phosphoryl group transfers, which is proposed to be substantial, will be revealed by these structural studies. %%% The goal of the proposed studies 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 will be 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. Two systems that utilize multi-phosphotransfer reactions have been selected: (a) A phosphoryl transfer chain that mediates sugar uptake across the membrane in bacteria. The three soluble proteins involved, Enzyme I, HPr and Enzyme IIA will be investigated. (b) The enzyme pyruvate phosphate dikinase that catalyses the inter-conversion of two high energy compounds, ATP and PEP, in some bacteria and plants. ***