STRUCTURE OF BRANCHED CHAIN KETO ACID DEHYDROGENASE

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Branched chain keto acid dehydrogenase is a multienzyme complex which is common to the metabolism of all three branched chain amino acids in pseudomonas and in mammals. In mammals it regulates the flow of metabolites from these amino acids into energy metabolism and into gluconeogenesis and is the site of the mutation in the cluster of inborn errors of metabolism known as maple syrup urine disease. Both the mammalian and bacterial complexes have been purified and are composed of four polypeptides, E1 (alpha and beta subunits), E2 and E3 (lipoamide dehydrogenase). The mammalian complex is regulated by phosphorylation of the E1 alpha subunit, the bacterial complex is regulated by L-valine which is a positive effector. The long-term objective of this research is to understand the structure, function and regulation of activity of the bacterial complex, which is a good model for understanding activity of both complexes. The Specific Aims of the present research are to: (1) Study the structure and function of the E1 and E2 subunits. The E1 subunits will be purified from E. coli TB1 carrying a plasmid with the structural genes for these subunits. These polypeptides will be characterized, used as antigens to prepare specific antisera and cyanogen bromide peptides will be partially sequenced. The DNA sequence of the structural gene will be determined in order to identify any evolutionary relationship to pyruvate and 2- ketoglutarate dehydrogenase of E. coli. A similar plan of study is proposed for the structure and function of the E2 subunit which will be compared with the E2 subunits of pyruvate and 2- ketoglutarate dehydrogenases of E. coli. (2) Study the regulation of gene expression of branched chain keto acid dehydrogenase operon. The objective of these studies will be to determine if the operon is positively or negatively regulated. The regulatory and structural genes will be separated by subcloning and the expression of the structural genes will be studied in the presence and absence of the regulatory gene. (3) Clone and sequence the pyruvate and 2-ketoglutarate dehydrogenase structural genes of P. putida. Both of these complexes will be cloned into pKT230, a broad host range plasmid, and the DNA sequence will be determined and compared with branched chain keto acid dehydrogenase as well as with pyruvate and 2-ketoglutarate dehydrogenase of E. coli.