) The concept of substrate channeling was originally put forth to explain the manner in which reactive intermediates are transferred from one protein to another in a metabolic pathway or shuttled from one active site to another within a single enzyme. Many have regarded the phenomenon as a critical method for the regulation of metabolic pathways within the living cell. The goal of this proposal is to explore, on a structural basis, the mechanisms of actions of the following enzymatic systems known to employ substrate channeling: carbamoyl phosphate synthetase, asparagine synthetase, and acetyl CoA carboxylase. The first two enzymes utilize substrate channeling between active sites within a single polypeptide chain while in the latter protein, the event occurs between active sites positioned on different polypeptide chains. These systems were selected for study not only because they use substrate channeling in their reaction mechanisms, but also because of their biological importance. Carbamoyl phosphate synthetase is involved in the de novo synthesis of pyrimidine nucleotides which are critical for cell proliferation. The enzyme also plays a key role in both arginine biosynthesis and the urea cycle in most terrestrial vertebrates. Asparagine synthetase catalyzes the ATP-dependent conversion of aspartic acid to asparagine and has been used in chemotherapeutic protocols for treating acute lymphoblastic leukemia. Finally, acetyl CoA carboxylase catalyzes the first committed and absolutely critical step in fatty acid synthesis. For these investigations, a combination of site-directed mutagenesis experiments and x-ray crystallographic analyses will be employed in order to more fully characterize the three-dimensional architectures of these proteins, their active site geometries, catalytic mechanismd, and the relationships of these parameters to substrate channeling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055513-07
Application #
6636228
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Jones, Warren
Project Start
1997-06-01
Project End
2005-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
7
Fiscal Year
2003
Total Cost
$291,000
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715