Hung Hui-Chih, Kuo Meng-Wei, Chang Gu-Gang, Liu Guang-Yaw
Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan, Republic of China.
Biochem J. 2005 Nov 15;392(Pt 1):39-45. doi: 10.1042/BJ20050641.
Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.
人类线粒体NAD(P)+依赖的苹果酸脱氢酶（脱羧）（苹果酸酶）可被富马酸特异性地变构激活。X射线晶体结构揭示了该酶在不存在和存在富马酸时的构象变化。先前的研究表明，富马酸通过精氨酸67和精氨酸91与变构口袋结合。这些残基的突变几乎消除了富马酸的激活作用。然而，这些氨基酸残基在一些不被富马酸激活的酶中是保守的，这表明可能存在其他控制激活机制的因素。在本研究中，我们试图阐明富马酸激活该酶的详细分子机制。定点诱变用于取代天冬氨酸102，它是富马酸结合口袋中的带电氨基酸之一，在其他脱羧苹果酸脱氢酶中不保守。为了探究该残基的电荷效应，将天冬氨酸102替换为丙氨酸、谷氨酸或赖氨酸。我们的实验数据清楚地表明天冬氨酸102对富马酸激活的重要性。将天冬氨酸102突变为丙氨酸或赖氨酸显著减弱了富马酸对该酶的激活作用。动力学参数表明，富马酸的作用主要是降低苹果酸、Mg2+和NAD+的米氏常数（Km）值，但并未显著提高催化常数（kcat）。随着富马酸浓度的增加，表观底物Km值降低。此外，富马酸激活的最大效应在低苹果酸、Mg2+或NAD+浓度下最为明显。随着苹果酸、Mg2+和NAD+浓度的增加，激活常数（Kact）值降低。然而，天冬氨酸102突变体对富马酸调节的敏感性要低得多。天冬氨酸102的突变导致富马酸与该酶底物之间协同效应的脱敏。
