STRUCTURAL DETERMINATION OF PURE

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 生化研究和基因组分析大肠杆菌和原鸡羧基氨基咪唑核糖核苷酸脱氢酶（PurE）表明，这些嘌呤生物合成酶提供了一个不寻常的例子，在一个高度保守的，主要的代谢途径的进化分歧。I类PurE，以E为代表。在大多数原核生物和真菌中发现的大肠杆菌蛋白，催化CO2基团从N5-羧基氨基咪唑核糖核苷酸（N5-CAIR）的氨基甲酸酯可逆转移到C4，产生4-羧基氨基咪唑核糖核苷酸（CAIR）。另一方面，以G.在高等真核生物中发现的鸡酶，通过CO2可逆转移到氨基咪唑核糖核苷酸（AIR）形成CAIR。 目前的结构研究涉及I类PurE（N5-羧基氨基咪唑变位酶），重点是其化学上独特的变位酶反应。一个工作机制的假设涉及组氨酸（His 45在大肠杆菌PurE）作为一个通用的酸，但没有证据表明多个质子化状态已获得。 结构的研究对于更好地理解其作用机理具有重要意义。 我们正在研究His 45突变体的结构。我们已经收集和解决的突变体之一的结构与CAIR和没有CAIR在1.75？2.1？分别 不对称单元由八聚体组成，电子密度图显示CAIR仅在两个活性位点结合。 我们也计划使用SSRL？的压力池做实验，了解CO2结合活性位点的特性。 研究突变酶、天然酶以及它们与高压CO2的复合物的晶体，可以使活性位点中的CO2结合位点定位成为可能。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Biochemical studies and genomic analyses of the Escherichia coli and Gallus gallus carboxyaminoimidazole ribonucleotide synthases (PurEs) indicate that these purine biosynthetic enzymes provide an unusual example of evolutionary divergence in a highly conserved, primary metabolic pathway. Class I PurEs, typified by the E. coli protein and found in most prokaryotes and fungi, catalyze the reversible transfer of a CO2 group from the carbamate of N5-carboxyamino imidazole ribonucleotide (N5-CAIR) to C4, yielding 4-carboxy aminoimidazole ribonucleotide (CAIR). On the other hand, class II PurEs, typified by the G. gallus enzyme and found in higher eukaryotes, form CAIR by reversible transfer of CO2 to aminoimidazole ribonucleotide (AIR). The current structural study involves class I PurE (N5-carboxyaminoimidazole mutase) with an emphasis on its chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. The structural study will be very important for getting a better understanding of the mechanism. We are studying the structures of the mutants of His45. We have collected and solved the structures of one of the mutants with CAIR and without CAIR at 1.75? and 2.1?, respectively. The asymmetric unit consists of an octamer and the electron density map shows binding of CAIR in two of the active sites only. We are also planning to use SSRL?s pressure cell to do experiment to understand CO2 binding characteristics of the active site. A study of the crystals of the mutant enzyme, native enzyme, and their complexes with high pressure CO2 may enable the location of CO2 binding sites in the active site.