Crystallographic Studies of Phosphoryl Group Transfer Reactions

9316934赫兹伯格将通过X射线结晶学确定参与磷基转移反应的蛋白质的三维结构，并将结合结晶学和定点突变方法研究反应机理。在上一次资助期间研究的磷酸烯醇式丙酮酸：蔗糖磷酸转移酶系统(PTS)将得到进一步研究。该项目还将包括另一种酶，丙酮酸磷酸二激酶(PPDK)，它显示了与PTS的酶I的序列同源性。PTS是一个多酶系统，通过磷酸基转移链调节糖的跨膜运输。在过去的两年里，我们已经确定了PTS的两个组分HPR和葡萄糖渗透酶的IIA结构域的晶体结构，并在原子水平上了解了它们之间的磷酰化转移反应的性质。这项拟议的工作将借鉴这些发现，并通过工程改变葡萄糖IIA结构域的磷酸化特异性来测试我们的理解。设计的突变体将进行生化分析以及X射线结晶学分析，以了解成功或失败的原因。PTS研究的第二部分是用MIR方法确定最近结晶的磷酸转移链上的第一个蛋白质-酶I的结构。这将为今后研究该系统的可溶性蛋白提供一个完整的结构基础。在结构确定之后，将进行抑制剂结合研究，以阐明磷酸烯醇式丙酮酸(PEP)的磷酸化和磷酸化转移到HPR的机制。第三部分完成了高盐和低盐形式的hpr的结构比较，以及hpr突变体H15A的结构提纯。这项工作对于解决目前关于hPR I S的磷酸化是否与骨架的构象转变有关的争议具有重要意义。我们的数据显示，情况并非如此。除了PTS的工作外，该项目的范围已经扩大到包括PPDK，这是一种催化PEP和ATP相互转化的多结构域酶。它与酶I的序列同源性来源于共同的底物PEP。PPDK总共催化了三个磷酰基转移，其中一个涉及焦磷酸中间体，这是酶促磷酰化转移反应中前所未有的中间体。共生梭菌酶结构的确定将有助于抑制物结合研究和诱变工作，以探索这种复杂酶的各种机制。这些结构研究将揭示与磷基转移相关的构象转变的性质，这被认为是实质性的。%拟议研究的目标是更好地了解蛋白质磷酸化调节的本质，蛋白质磷酸化反应是广泛的生物过程的中心。将结合X射线结晶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突变方法来探索它们的功能。已经选择了两个利用多磷转移反应的系统：(A)在细菌中介导糖跨膜吸收的磷酰转移链。涉及的三种可溶性蛋白，酶I，HPR和酶IIA将被研究。(B)在一些细菌和植物中催化两种高能化合物ATP和PEP相互转化的丙酮酸磷酸二激酶的酶。***

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. ***