The overall objective is to establish the mechanism responsible for regulation of the oxidation of branched chain amino acids by mammalian cells. The work will focus on the branch chain alpha-ketoacid dehydrogenase complex (BCKDH), well established as a regulatory enzyme for branched chain amino acid catabolism. Studies will be conducted with purified BCKDH, its kinase, its phosphatase, H4IIEC3 hepatoma cells, fibroblasts, and intact animals. The working hypothesis is that BCKDH is regulated by covalent modification dictated in the short term by the sensitivity of BCKDH kinase to inhibition by alpha-ketoiso- caproate and in the long term by alterations in BCKDH kinase activity. This work will explain the basis for alteration in total BCKDH kinase activity, explain the basis for defective branched chain amino acid catabolism in the diabetic state, elucidate the role of the individual phosphorylation sites of the BCKDH-E1alpha subunit in regulating enzyme activity, and provide structure-function information about the E1 component of the complex. The detailed specific aims are: (a) to define the factors responsible for regulation of the in vivo capacity of individual tissues to oxidize branched chain amino acids; (b) to characterize BCKDH kinase, and to establish the mechanism responsible for nutritional/hormonal alterations in the activity/mass of BCKDH kinase; (c) to establish the basis for site specificity of BCKDH kinase and the relative importance of the individual phosphorylation sites of the E1alpha subunit in the regulation of BCKDH activity; and (d) to define structural features of the E1 component important to its function in the BCKDH complex. These studies should further our understanding of altered branched chain amino acid catabolism known to occur in numerous diseases and metabolic conditions, including diabetes, obesity, kwashiorkor, and hepatic encephalopathy. They should also contribute to the development of methods for prenatal diagnosis and detection of carriers of maple syrup urine disease.
| Shimomura, Yoshiharu; Honda, Takashi; Shiraki, Makoto et al. (2006) Branched-chain amino acid catabolism in exercise and liver disease. J Nutr 136:250S-3S |
| Joshi, Mandar A; Jeoung, Nam Ho; Obayashi, Mariko et al. (2006) Impaired growth and neurological abnormalities in branched-chain alpha-keto acid dehydrogenase kinase-deficient mice. Biochem J 400:153-62 |
| Harris, Robert A; Joshi, Mandar; Jeoung, Nam Ho et al. (2005) Overview of the molecular and biochemical basis of branched-chain amino acid catabolism. J Nutr 135:1527S-30S |
| Harris, Robert A; Joshi, Mandar; Jeoung, Nam Ho (2004) Mechanisms responsible for regulation of branched-chain amino acid catabolism. Biochem Biophys Res Commun 313:391-6 |
| Obayashi, Mariko; Shimomura, Yoshiharu; Nakai, Naoya et al. (2004) Estrogen controls branched-chain amino acid catabolism in female rats. J Nutr 134:2628-33 |
| Kobayashi, Rumi; Murakami, Taro; Obayashi, Mariko et al. (2002) Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms. Arch Biochem Biophys 407:231-40 |
| Harris, R A; Kobayashi, R; Murakami, T et al. (2001) Regulation of branched-chain alpha-keto acid dehydrogenase kinase expression in rat liver. J Nutr 131:841S-845S |
| Obayashi, M; Sato, Y; Harris, R A et al. (2001) Regulation of the activity of branched-chain 2-oxo acid dehydrogenase (BCODH) complex by binding BCODH kinase. FEBS Lett 491:50-4 |
| Baker, L J; Dorocke, J A; Harris, R A et al. (2001) The crystal structure of yeast thiamin pyrophosphokinase. Structure 9:539-46 |
| Timm, D E; Liu, J; Baker, L J et al. (2001) Crystal structure of thiamin pyrophosphokinase. J Mol Biol 310:195-204 |
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