| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 94 === Human mitochondrial NAD(P)+-dependent malic enzyme (EC1.1.1.39, HME) catalyzes the oxidative decarboxylation of malate to pyruvate and carbon dioxide with the concomitant reduction of NAD(P)+ to NAD(P)H. The enzyme is a tetramer composed of double dimer structure. Malic enzyme has two interfaces, the dimer and tetramer interface with different interactions. To investigate the amino acid involved in the different interface, we used different pH buffers to alter the ionization states of amino acid side chains. The enzyme structure changes in different pH buffers were analyed with circular dichroism, fluorescence and analytical ultra - centrifugation. We found that HME underwent reversible dissociation from neutral pH to pH 3.88, further decreasing pH induced unfolded. In the pH between 3.88 and 3.58, HME existed as molten globule state, which was detected by a molecular probe, 1-anilinonaphthalene-8-sulfonic acid that registered enzyme unfolding because of the intrinsic hydrophobic regions exposure. In the refolding experiments, the enzyme dissociated at pH 4.45 was reassociated to tetramer when the solution was titrated back to neutral pH. From these reversible dissociation data, the dissociation constants of dimer and tetramer interfaces, were determined to be 5.8 and 5.2, and 6.0 and 5.7, respectively. The protein aggregation at pH < 3.34 was irreversible. Inclusion of Mg2+ divalent metal ion stabilized the quaternary structure of HME against acid induced conformational change.
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