MECHANISM & STRUCTURE OF HUMAN GAR TRANSFORMYLASE

这项建议描述了对人类依赖叶酸的从头开始的嘌呤生物合成酶甘氨酰胺核糖核苷酸转移酶的机制和结构的多方面研究。在高等真核生物中，这种活性构成了单体三功能蛋白的C-末端结构域，它还催化甘氨酰胺核苷酸的合成和氨基咪唑核苷酸的合成。甘氨酰胺核糖核苷酸转移酶的活性已被证明是癌症化疗的有效靶点。该计划的总体目标是：确定转化反应的动力学和化学机制；确定参与底物结合和催化的蛋白质残基；确定甘氨酰胺核糖核酸结构域和三功能酶的结构；检查甘氨酰胺核糖核苷酸转换酶和甘氨酰胺核糖核苷酸合成酶结构域是否相互作用并确定这种潜在相互作用的功能后果。为此，描述了一系列的动力学研究、定点突变研究、蛋白质-蛋白质相互作用研究、底物通道研究和X射线结晶学研究。这些研究将提供关于人类转移酶的迫切需要的机制和结构信息，并将确定由结构域相互作用引起的底物通道是否与该系统有关。这些研究还将为今后研究构成从头合成嘌呤生物合成途径的酶的相互作用、调节该途径的机制(S)以及设计人转甲酰基酶的特异性抑制剂奠定基础。虽然已经证明抑制这一途径有可能用于癌症化疗，但关于该途径及其组成酶的许多基本信息仍有待阐明。

This proposal describes a multi-faceted investigation of the mechanism and structure of the human folate-dependent, de novo purine biosynthetic enzyme, glycinamide ribonucleotide transformylase. In higher eukaryotes, this activity constitutes the C-terminal domain of a monomeric trifunctional protein which also catalyzes the synthesis of glycinamide ribonucleotide and the synthesis of aminoimidazole ribonucleotide. The glycinamide ribonucleotide transformylase activity has been demonstrated to be an effective target for cancer chemotherapy. The overall goals of this program are to: determine the kinetic and chemical mechanisms of the transformylation reaction; identify the protein residues involved in substrate binding and catalysis; determine the structures of the glycinamide ribonucleotide domain and the trifunctional enzyme; and examine whether the glycinamide ribonucleotide transformylase and the glycinamide ribonucleotide synthetase domains interact and define the functional consequences of this potential interaction. To these ends, a series of kinetic studies, site-directed mutagenesis studies, protein-protein interaction studies, substrate channeling studies, and x-ray crystallographic studies are described. These studies will afford much needed mechanistic and structural information about the human transformylase and will establish whether substrate channeling resulting from domain interactions pertains in this system. These studies will also provide a foundation for future studies on the interactions of the enzymes which constitute the de novo purine biosynthetic pathway, the mechanism(s) of regulation of the pathway, and the design of specific inhibitors for the human trans-formylase. Although it has been demonstrated that inhibition of this pathway shows potential for cancer chemotherapy, much basic information about the pathway and its constituent enzymes remains to be elucidated.