REGULATION OF THIAMINE DEPENDENT ENZYMES INVOLVED IN GLUCOSE METABOLISM

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Because of their importance in the energy and intermediary metabolism, we studied the biochemical and molecular characteristics of thiamine- dependent enzymes [the mitochondrial pyruvate dehydrogenase (PDH) complex, the mitochondrial `-ketoglutarate dehydrogenase (`-KGDH) complex, and cytosolic transketolase (TK).] These enzymes play key roles in glucose metabolism which is critically important in cerebral energy supply. Reduced activities of these thiamine-dependent enzymes were reportedly observed in many neurodegenerative disease states including alcoholic Wernicke-Korsakoff patients. Catalytically active PDH E1 proteins of human and rat were over-produced in E. coli. Site-directed mutagenesis of the E1 subunit simulating naturally occurring mutations were performed to study the molecular mechanism of how the structural mutations affect the catalytic activity or the stability of the PDH E1 subunit. Methods to purify the over-produced proteins to near homogeneity were developed: cell homogenation, ammonium sulfate precipitation, and chromatography on phenyl-sepharose column connected to FPLC. We then examined the biochemical properties of the purified human PDH E1 proteins of both wild and mutant PDH E1 proteins over-produced in E. coli. Differential rate of protein phosphorylation was observed in the mutant PDH protein, which may correspond for the rapid loss of the activity in this mutant. In addition, DNA polymorphism in the human PDH gene is being studied by techniques of reverse transcription-polymerase chain reaction (RT-PCR) followed by analyses of single strand conformational polymorphism (SSCP). The critical role of arginine residue in the catalytic activity of transketolase was studied with the aid of site-directed mutagenesis technique. Among the four conservative arginine residues in transketolase (amino acids 62, 359, 455, and 528), only Arg359 appears to be essential for the TK activity. Computer- assisted homology modelling of the PDH E1 is being accomplished using the crystal structure of yeast transketolase obtained from the Swiss Protein Data Bank.