The long-term goal of this project is to understand the structure and function of a novel family of mitochondrial protein kinases (mPKs). The mPK members consist of branched-chain alpha-ketoacid dehydrogenase kinase (BCK) and the four isoforms of pyruvate dehydrogenase kinase. They are molecular switches that down-regulate the oxidation of alpha-ketoacids and pyruvate. Elevated levels of these metabolites are implicated in disease states such as insulin-resistant Type II diabetes, branched-chain ketoaciduria, and primary lactic acidosis. BCK is a component of the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. This macromolecular multi-enzyme complex is organized about a 24-meric transacylase (E2b) scaffold, to which a decarboxylase (Elb), a dehydrogenase (E3), the BCK and the BCKD phosphatase are attached through ionic interactions. The P.I.'s laboratory has recently determined the structure of the rat BCK. The BCK structure features a characteristic nucleotide-binding domain and a four-helix bundle domain. These two domains are reminiscent of modules found in protein histidine kinases (PHKs), which are involved in two-component signal transduction systems. In this application, the P.I. proposes: 1) to identify and characterize the domains/regions in BCK, which interact with Elb (the substrate) and E2b (the regulator) components of the BCKD complex; 2) to decipher the functional significance of nucleotide-induced domain communication in BCK; 3) to elucidate the reaction mechanisms for the BCK-catalyzed ATP hydrolysis and phosphotransfer in BCK. The information derived from the proposed studies will provide mechanistic insights into the regulation of alpha-ketoacid dehydrogenase complexes by reversible phosphorylation. This knowledge will have wide implications for understanding conservation in the reaction mechanism for protein kinases involved in signal transduction as well as how this mechanism is perturbed in human diseases.
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