Structure and Function of Human Mitochondrial NAD(P)+-Dependent Malic Enzyme: Effects of Exo Site Residues

• Main Authors: Wan-Ting Shih, 石婉婷
• Other Authors: Hui-Chih Hung
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/52424661831251174226

碩士 === 國立中興大學 === 生命科學系所 === 102 === Malic enzyme (ME) requires the presence of divalent metal ion as cofactor to catalyze a reversible oxidative decarboxylation of L-malate to yield pyruvate and CO2 with the reduction of NAD(P)+ into NAD(P)H. In mamamals, malic enzymes have been classified three isoforms according to their cofactor specifity and subcellular localization as following: cytosolic NADP-dependent (c-NADP-ME, ME1), mitochondrial NADP -dependent (m-NADP-ME, ME2), and mitochondrial NAD(P) -dependent (m-NAD(P)-ME, ME2). Although crystal structure and sequence of ME1and ME2 are similar, the regulatory mechanisms are distinct. From crystal structure, the NAD+ and ATP competively bind to the active sites and exo sites of each monomer. We aim to understand the role of NAD+ and ATP in regulating the structure of function of the ME2. To determine the enzyme kinetic and structure stability of the exo site in ME2. The mutagenesis experiment was used to examine the functional amino acid residues, a series of mutants corresponding to ME1 were generated including R144A, H154V, K156A, G192A, R194N, R197E, D244S, R245K, I479V, N482G, R542V, Y552F, R556Q. The activity thermal stability, and quantility structure of ME2 with NAD+ or ATP binding were determine by the spectrometer, circular dichroism, differential scanning calorimeter, analytical ultracentrifuge. In conclusion, the affinity of ligand to exo siteinflence the structural stability of ME2. More importantly, Arg197, Arg542, and Arg556 of exo site are critical for ATP binding and stabilization of exo site structure.