In biological systems, most processes are under the control of a variety of regulatory mechanisms. For example, the glyoxylate bypass of Escherichia coli is controlled by the phosphorylation of isocitrate dehydrogenase (IDH), by regulation of the expression of the glyoxylate bypass operon and by allosteric mechanisms. Our long term goal is to characterize the functioning and coordination of these regulatory mechanisms. This proposal focuses on the structure/function relationships of IDH kinase and IDH phosphatase and the regulation of the phosphorylation cycle in vivo. IDH kinase and phosphatase have been found to be physically associated, probably on the same polypeptide chain. In an effort to determine the regulatory and structural significance of this bifunctional structure, the relationships between the functional domains will be characterized. In addition to the methods of protein chemistry, this study will make extensive use of recombinant DNA technology, including mutagenesis of the gene encoding IDH kinase/phosphatase. Specific problems to be addressed include the location of these domains, the degree to which they are physically autonomous and the potential for interdomain allosteric communication. During the analysis of the structure of IDH kinase/phosphatase, a variety of normal and mutant clones of the gene encoding this protein are being constructed. These clones provide an ideal approach to the study of the IDH phosphorylation cycle in vivo. The cellular level and properties of IDH kinase/phosphatase will be manipulated using these clones and the effects of these perturbations will be characterized. The results of these experiments will provide the basis for the construction of models which describe the regulation of IDH phosphorylation in the intact organism.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033927-07
Application #
3284120
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1984-07-01
Project End
1991-08-31
Budget Start
1990-07-01
Budget End
1991-08-31
Support Year
7
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Ikeda, T P; Houtz, E; LaPorte, D C (1992) Isocitrate dehydrogenase kinase/phosphatase: identification of mutations which selectively inhibit phosphatase activity. J Bacteriol 174:1414-6
Ikeda, T; LaPorte, D C (1991) Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity. J Bacteriol 173:1801-6
Resnik, E; LaPorte, D C (1991) Introduction of single-copy sequences into the chromosome of Escherichia coli: application to gene and operon fusions. Gene 107:19-25
Laporte, D C; Stueland, C S; Ikeda, T P (1989) Isocitrate dehydrogenase kinase/phosphatase. Biochimie 71:1051-7
Stueland, C S; Ikeda, T P; LaPorte, D C (1989) Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase. J Biol Chem 264:13775-9
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Chung, T; Klumpp, D J; LaPorte, D C (1988) Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map. J Bacteriol 170:386-92
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Stueland, C S; Eck, K R; Stieglbauer, K T et al. (1987) Isocitrate dehydrogenase kinase/phosphatase exhibits an intrinsic adenosine triphosphatase activity. J Biol Chem 262:16095-9