We are studying the enzyme, formyltetrahydrofolate synthetase, to examine a number of problems of interest to enzymologists; mechanism, substrate binding and control of stucture by small ligands. The experiments outlined are designed to increase our understanding of the relationship between structure and catalytic activity of this enzyme and thereby add to our understanding of enzymes in general. We plan to use 13C, 1H and 31P NMR relaxation techniques to study ligand binding. The studies will be done under different conditions to determine internuclear distances between the nucleus and enzyme-bound Mn(II); whether detectable differences exist in the binding sites of the monomer and tetramer, and whether conformational changes are produced upon ligand binding. UV-difference and fluorescence spectroscopy will be used to study ATP and ATP-analog interactions with the enzyme. Various experiments are proposed to find evidence for the existence of an enzyme-bound formyl phosphate as an intermediate. The binding site for ATP will be explored by using UV-induced crosslinking of photo-activatable ATP analogs, followed by proteolytic digestion. Studies of the cation-induced monomer reassociation reaction are also proposed. The assembly mechanism will be followed by UV-difference spectroscopy and conventional and rapid kinetic techniques. In some of the experiments comparisons will be made between the clostridial enzyme, the primary protein being studied, and the yeast enzyme. Major structural differences exist between the procaryotic and eucaryotic enzyme, which makes these comparisons of interest.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK007140-26
Application #
3224511
Study Section
Biochemistry Study Section (BIO)
Project Start
1979-04-01
Project End
1994-03-31
Budget Start
1991-09-01
Budget End
1994-03-31
Support Year
26
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Kansas Lawrence
Department
Type
Schools of Arts and Sciences
DUNS #
072933393
City
Lawrence
State
KS
Country
United States
Zip Code
66045