MECHANISM & STRUCTURE OF HUMAN GAR TRANSFORMYLASE

这一建议描述了一个多方面的研究机制和结构的人叶酸依赖，新的嘌呤生物合成酶，甘氨酸酰胺核糖核苷酸转化酶。在高等真核生物中，该活性构成一个单体三功能蛋白的c端结构域，该结构域还催化甘氨酸酰胺核糖核苷酸和氨基咪唑核糖核苷酸的合成。甘氨酸酰胺核糖核苷酸转化酶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；鉴定参与底物结合和催化的蛋白质残基；确定甘氨酸酰胺核糖核苷酸结构域和三功能酶的结构；并检查甘氨酸酰胺核糖核苷酸转化酶和甘氨酸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。为了达到这些目的，本文描述了一系列动力学研究、定点诱变研究、蛋白质-蛋白质相互作用研究、底物通道研究和x射线晶体学研究。这些研究将提供关于人类转化酶的机制和结构信息，并将确定由结构域相互作用引起的底物通道是否属于该系统。这些研究也将为未来研究构成从头嘌呤生物合成途径的酶的相互作用、该途径的调节机制以及设计人类反式甲酰基酶特异性抑制剂提供基础。虽然已经证明抑制这一途径具有癌症化疗的潜力，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.