The overall objective of this proposal is to clarify poorly understood aspects of the regulation of leucine catabolism by mammalian tissues. The work is focused on the regulation of the branched chain alpha-Ketoacid dehydrogenase (BCKDH) complex by its kinase and phosphatase. Proposed experiments are designed to answer four major questions: 1. What amin acid residues are involved in E1 binding of thiamin pyrophosphate (TPP), the essential cofactor for this component of the BCKDH complex? 2. What is the mechanism by which phosphorylation inhibits the dehydrogenase activity of BCKD E1? 3. How does BCKDH kinase recognize the site (E1-alpha-Ser293) that it phosphorylates on BCKDH E1? 4. What are the characteristics of the BCKDH phosphatase, and how is its activity toward the BCKDH complex regulated? The working hypotheses that will guide the work include: 1. A model based on the three-dimensional structure of transketolase identifies amino acid residues that may be involved in the binding of TPP by BCKDH E1; 2. Phosphorylation of E1-alpha-Ser293 by BCKDH kinase inactivates E1 by inhibiting TPP binding; and 3. The specificity of BCKDH kinase depends both on the amino acid residues spatially located in the vicinity of the E1 phosphorylation site and on the positioning of the kinase by E2 binding. The detailed specific aims are: 1. To establish amino acid residues critical to cofactor binding by the E1 component of the BCKDH complex; 2. To establish the molecular mechanism responsible for inhibition of BCKDH E1 activity by phosphorylation; 3. To establish the molecular basis for recognition of E1-alpha phosphorylation sites by BCKDH kinase; and 4. To clone, express, and characterize BCKDH phosphatase and determine its role in regulation of the activity state of the BCKDH complex.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK019259-17A2
Application #
2611615
Study Section
Biochemistry Study Section (BIO)
Program Officer
Laughlin, Maren R
Project Start
1978-07-01
Project End
2002-03-31
Budget Start
1998-04-10
Budget End
1999-03-31
Support Year
17
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Biochemistry
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
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