EFFECTOR BINDING SITES AND REGULATORY MECHNISIMS IN PEP CARBOXYLASE

磷酸烯醇式丙酮酸(PEP)羧基酶服务于多种代谢 细菌和植物的功能，包括在C4中的中心作用 光合作用途径。而推导出的该酶的氨基酸序列 从几个来源可以获得，关于位置或 这种酶的变构部位的组成。这样做的总体目标是 该项目利用亲和标记和定点突变来 促进我们对效应部位和调控机制的理解 这种酶。该项目的具体目标包括：1) 竞争我们当前项目中的剩余工作，以分离和排序 来自已被磷酸化的激活剂结合部位的肽 用高碘酸氧化型AMP(OAMP)2标记)探针定位 腺苷三磷酸与三磷酸腺苷抑制部位的特异性及其潜在的作用 亲和标记物oATP3)比较 两个玉米PEP羧化酶全长克隆pGLW5的重组 和pGLW6，并提高这两种PEP的表达水平 大肠杆菌中的羧基酶克隆使用定点突变来探测 被怀疑具有调节作用的残基的功能。 目标残基包括苏氨酸227和几个残基 在这一残基附近的区域，在高等植物和 细菌PEP羧基酶，已知对激活至关重要 该酶由1，6-二磷酸果糖组成。其他目标残留物包括 通过与OAMP的亲和标记鉴定多肽中的那些，以及 最终，通过oATP标记来鉴定那些。信息来自 这些研究将提供对腺苷和糖磷酸盐的洞察。 这种酶的调节位点，更广泛地说，进入到 在酶水平上的代谢调节。

Phosphoenolpyruvate (PEP) carboxylase serves a number of metabolic functions in bacteria and plants, including a central role in the C4 photosynthetic pathway. While deduced amino acid sequences of the enzyme from several sources are available, little is known about the location or composition of the allosteric sites of this enzyme. The broad goal of this project is to utilize affinity labeling and site-directed mutagenesis to advance our understanding of effector sites and regulatory mechanisms of this enzyme. The specific aims of the project include the following: 1) Compete the remaining work in our current project to isolate and sequence the peptide from the phosphorylated activator binding site which has been labeled with periodate-oxidized AMP (oAMP) 2) Probe the location and specificity of the ATP inhibition site with oAMP and the potential affinity label oATP 3) Compare the physical and kinetic properties of recombinant maize PEP carboxylase from two full length cDNA clones, pGLW5 and pGLW6, and improve the level of expression of these two PEP carboxylase clones in E. coli 4) Use site-directed mutagenesis to probe the function of residues that are suspected of having a regulatory role. The target residues include threonine 227 and several residues in the region near this residue, which is conserved among higher plant and bacterial PEP carboxylases and which is known to be crucial for activation of the enzyme by fructose 1 ,6 bisphosphate. Other target residues are those in the peptide being identified by affinity labeling with oAMP, and ultimately those identified by labeling with oATP. The information from these studies will provide insight into the adenylate and sugar phosphate regulatory sites of this enzyme and, more generally, into the process of metabolic regulation at the enzyme level.