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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM061194-01
Application #
6090307
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Ikeda, Richard A
Project Start
2000-04-01
Project End
2004-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
1
Fiscal Year
2000
Total Cost
$258,478
Indirect Cost
Name
University of Cincinnati
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Manieri, Wanda; Moore, Molly E; Soellner, Matthew B et al. (2007) Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes. Biochemistry 46:156-63