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

Autor: Chien-Hui Hsu, 徐千惠
Rok vydání: 2014
Druh dokumentu: 學位論文 ; thesis
Popis: 102
Malic enzyme (ME) is a homotetrameric enzyme, which amino acid sequences are highly conserved in all the species. In mammalians, ME can be divided into three isoforms according to their subcellular distributions and coenzyme specificities：a cytosolic NADP+-dependent isoform (c-NADP-ME, ME1), mitochondrial NAD(P)dependent isoform (m-NAD-ME, ME2), and mitochondrial NADP+-dependent isoform (m-NADP-ME, ME3). Different from human c-NADP-ME, m-NAD(P)-ME have an allosteric site for fumarate binding to regulate enzyme activity, as its activator. Furthermore, on the basis of structure organization, m-NAD(P) -ME can be distinguished into four different forms, open forms Ⅰ and II, and close forms Ⅰ and II. The binding of either malate or fumarate may induce the transition from open form I to open form II, and induce the fumarate binding site formation and abolished the malate binding cooperativity in close form II. In order to investigate the non-conserved residues of fumarate binding site in human m-NAD(P)-ME and c-NADP-ME, a series of site-directed mutagenesis were created to measure the enzyme activity. By classification, the first group of A65V and E90D abolished fumarate activation and didn’t affect enzyme activity; the second group of I88L and G124S abolished fumarate activation and decreased enzyme activity; the third group of E59N apparently inhibited by fumarate; the fourth group of K57S、L79F、I83L and Y175N had over-activation by fumarate ; the last group of K74H and G87D had fumarate activation but decreased enzyme activity. By using circular dichroism spectroscopy (CD), spectrofluorometer, differential scanning calorimeter (DSC), the stability of secondary structure, tertiary structure and full protein in m-NAD(P)-ME mutants were analyzed to understand the effects of fumarate binding site residues on enzyme activity and structure stability.
Databáze: Networked Digital Library of Theses & Dissertations
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