? The activity of mammalian pyruvate dehydrogenase complex is regulated through the interconversion of phosphorylated (inactive) and dephosphorylated (active) forms. This regulation is of great importance to the control of the metabolic fate of pyruvate and carbohydrate fuels. Activation of the complex by dephosphorylation increases the availability of acetyl-CoA for complete oxidation by the citric acid cycle in peripheral tissues and for fatty acid synthesis in the liver. Inactivation of the complex by phosphorylation limits the use of glucose by peripheral tissues and conserves three carbon compounds for glucose synthesis by the liver and kidney. Importantly, the inactivation of pyruvate dehydrogenase complex also occurs in diabetes. This exacerbates the diabetic state by inappropriately sparing pyruvate in the face of abundant levels of blood glucose. The long-term goal of this project is to understand the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase activity under normal circumstances as well as in diabetes. The key role in this regulation belongs to pyruvate dehydrogenase kinase. This kinase is an integral part of the complex, tightly associated with the transacetylase component. It is generally believed that due to this tight association, the kinase functionality is largely determined by protein-protein interactions between kinase and lipoyl-bearing domains located on the transacetylase component of the complex. In this application, we propose to test this hypothesis using a combination of X-ray crystallography and structure/function analysis.
The Specific Aims for this proposal are: 1) to determine the three-dimensional structure of the kinase molecule in a complex with the inner lipoyl-bearing domain; 2) to identify the molecular features determining the specificity of recognition of lipoyl-bearing domains by different kinase isozymes; 3) to establish the molecular mechanism(s) responsible for the lipoyl-bearing domain-mediated regulation of kinase activity; and 4) to elucidate the mechanism(s) of action of novel kinase-specific inhibitors that are thought to be acting through the lipoyl-bearing domain binding site of kinase molecule. The structural information generated in the course of this study will be indispensable for the design of a new generation of kinase-specific drags. These compounds may prove to be beneficial in alleviating some of the symptoms associated with diabetes, as well as metabolic acidosis, ischemic heart disease, and sepsis. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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Ikeda, Richard A
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University of Alabama Birmingham
Schools of Medicine
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Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M (2008) Allosteric coupling in pyruvate dehydrogenase kinase 2. Biochemistry 47:8358-66
Tuganova, Alina; Klyuyeva, Alla; Popov, Kirill M (2007) Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2. Biochemistry 46:8592-602
Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M (2007) Amino acid residues responsible for the recognition of dichloroacetate by pyruvate dehydrogenase kinase 2. FEBS Lett 581:2988-92
Tuganova, Alina; Popov, Kirill M (2005) Role of protein-protein interactions in the regulation of pyruvate dehydrogenase kinase activity. Biochem J 387:147-53
Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M (2005) The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity. Biochemistry 44:13573-82
Karpova, Tatiana; Danchuk, Svitlana; Huang, Boli et al. (2004) Probing a putative active site of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 (PDP1c) by site-directed mutagenesis. Biochim Biophys Acta 1700:43-51
Burelle, Yan; Wambolt, Richard B; Grist, Mark et al. (2004) Regular exercise is associated with a protective metabolic phenotype in the rat heart. Am J Physiol Heart Circ Physiol 287:H1055-63
Karpova, Tatiana; Danchuk, Svitlana; Kolobova, Elena et al. (2003) Characterization of the isozymes of pyruvate dehydrogenase phosphatase: implications for the regulation of pyruvate dehydrogenase activity. Biochim Biophys Acta 1652:126-35
Boulatnikov, Igor; Popov, Kirill M (2003) Formation of functional heterodimers by isozymes 1 and 2 of pyruvate dehydrogenase kinase. Biochim Biophys Acta 1645:183-92
Tuganova, Alina; Boulatnikov, Igor; Popov, Kirill M (2002) Interaction between the individual isoenzymes of pyruvate dehydrogenase kinase and the inner lipoyl-bearing domain of transacetylase component of pyruvate dehydrogenase complex. Biochem J 366:129-36

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