EFFECTOR BINDING SITES AND REGULATORY MECHNISIMS IN PEP CARBOXYLASE

磷酸烯醇式丙酮酸（PEP）羧化酶是一种重要的代谢酶。 在细菌和植物中的功能，包括在C4 光合途径同时推导出该酶的氨基酸序列 从几个来源是可用的，很少有人知道的位置或 该酶的变构位点的组成。这个广泛的目标 项目是利用亲和标记和定点突变， 推进我们对效应位点和调控机制的理解， 这种酶。该项目的具体目标包括：1） 完成当前项目中的剩余工作，以进行隔离和排序 来自磷酸化激活剂结合位点的肽，其已经被 用高碘酸盐氧化的AMP（oAMP）标记2）探测位置， ATP抑制位点与oAMP的特异性和潜在的 3）比较亲和标记oATP的物理和动力学性质， 从两个全长cDNA克隆pGLW 5中获得的重组玉米PEP羧化酶 和pGLW 6，并提高这两种PEP的表达水平 羧化酶克隆E. 4）使用定点突变来探测 被怀疑具有调节作用的残基的功能。 靶残基包括苏氨酸227和多核苷酸中的几个残基。 在这个残基附近的区域，这是高等植物中保守的， 细菌PEP羧化酶，并且已知其对于活化至关重要 果糖1，6-二磷酸对酶的作用其他靶残基是 肽中的那些通过用oAMP亲和标记来鉴定，和 最终通过oATP标记鉴定的那些。的信息 这些研究将提供深入了解腺苷酸和糖磷酸 这种酶的调节位点，更一般地说，进入过程， 酶水平的代谢调节。

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.