The mammalian branched chain alpha-ketoacid dehydrogenase complex: I. Regulation by branced chain alpha-ketoacid dehydrogenase kinase. II. Defects in the E1alpha, E1beta and E2 subunits

The branched chain α-ketoacid dehydrogenase (BCKD) multienzyme complex catalyzes the first irreversible step in the catabolism of the branched chain amino acids (BCAA). This mitochondrial complex consists of four catalytic components designated E1α, E1β, E2, and E3, and two regulatory components. The regulatory components include a specific kinase that phosphorylates and inactivates the BCKD complex, and a specific phosphatase that dephosphorylates and activates BCKD. This work focuses on the regulation of BCKD and defects in its components, both of which can alter BCAA metabolism.; Phosphorylation/dephosphorylation is the predominant mechanism of regulation of BCAA catabolism. The proportion of BCKD that is active (unphosphorylated) is inversely related to the expression of BCKD-kinase, which varies in a tissue-specific manner and fluctuates with diet, exercise, and hormonal stimuli. Here, the effects of the anti-catabolic hormone insulin on the expression of BCKD-kinase was examined. Insulin was found to upregulate BCKD-kinase expression and cause a corresponding decrease in the active portion of BCKD through post-transcriptional mechanisms. This BCKD-kinase induction was found to be mediated via signaling pathways involving PI 3-kinase and mTOR. These findings suggest that insulin is important in regulating BCAA catabolism.; Inherited defects in the catalytic subunits of BCKD prevent the catabolism of the BCAA and result in M&barbelow;aple S&barbelow;yrup U&barbelow;rine D&barbelow;isease. Finding the defective subunit and defining the mutations in MSUD patients could provide information for optimization of their treatment. In a second project, an enzyme complementation method utilizing retroviral gene transfer was established to identify the mutant gene in MSUD patient cell lines. Results obtained from 63 cell lines showed that 31% of the patients had mutations in E1α, 38% in E1β, and 19% in E2. Nucleotide changes were determined in a subset of these patients and 9 previously unreported mutations were found. Finally, clinical characteristics were compared between patients with mutations in each gene. No significant differences in clinical presentation were found between patients with mutations in E1α, E1β, or E2. The information obtained here about patient mutations will lead to a better understanding of the structure and function of BCKD and will be useful for developing new therapies for MSUD.