| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 89 === Pigeon liver malic enzyme [EC1.1.1.40] is a homotetramer with each subunit of molecular weight 62 kDa. This enzyme is a bifunctional enzyme that catalyzes the reversible oxidative decarboxylation of L-malate, yielding pyruvate and CO2, with a concomitant conversion of coenzyme NADP+ to NADPH. The three dimensional structures of pigeon malic enzyme was constructed by the computer modeling. There are six threonine residues closed to the active site. These six threonine residues were mutated to alanine residues by alanine scanning site-directed mutagenesis. Mutation of threonine residues at positions 92 and 399 showed significant effect on the Km value for malate. Replacement of these two threonine residues to valine or serine and the double mutant T(92,399)A were constructed. Initial velocity studies demonstrated that both hydroxyl group and correct carbon chain are essential for malate binding. Inhibition studies of malate analogs, DL-a-hydroxybutyric acid and DL-b-hydroxybutyric acid, were also performed. In conclusion, we suggest that Thr 92 may interact with C4 carboxyl group of malate through a hydrogen bond.
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