Dataset Information

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme).

ABSTRACT: 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.

SUBMITTER: Hung HC

PROVIDER: S-EPMC1317662 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme).

Hung Hui-Chih HC Kuo Meng-Wei MW Chang Gu-Gang GG Liu Guang-Yaw GY

The Biochemical journal 20051101 Pt 1

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 con ...[more]

PMID: 15989682

Similar Datasets

Project description:Oxygenic photoautotrophic bacteria, cyanobacteria, have the tricarboxylic acid (TCA) cycle, and metabolite production using the cyanobacterial TCA cycle has been spotlighted recently. The unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) has been used in various studies on the cyanobacterial TCA cycle. Malate oxidation in the TCA cycle is generally catalyzed by malate dehydrogenase (MDH). However, Synechocystis 6803 MDH (SyMDH) is less active than MDHs from other organisms. Additionally, SyMDH uses only NAD+ as a coenzyme, unlike other TCA cycle enzymes from Synechocystis 6803 that use NADP+. These results suggest that MDH rarely catalyzes malate oxidation in the cyanobacterial TCA cycle. Another enzyme catalyzing malate oxidation is malic enzyme (ME). We clarified which enzyme oxidizes malate that originates from the cyanobacterial TCA cycle using analyses focusing on ME and MDH. In contrast to SyMDH, Synechocystis 6803 ME (SyME) showed high activity when NADP+ was used as a coenzyme. Unlike the Synechocystis 6803 mutant lacking SyMDH, the mutant lacking SyME accumulated malate in the cells. ME was more highly preserved in the cyanobacterial genomes than MDH. These results indicate that ME mainly oxidizes malate that originates from the cyanobacterial TCA cycle (named the ME-dependent TCA cycle). The ME-dependent TCA cycle generates NADPH, not NADH. This is consistent with previous reports that NADPH is an electron carrier in the cyanobacterial respiratory chain. Our finding suggests the diversity of enzymes involved in the TCA cycle in the organisms, and analyses such as those performed in this study are necessary to determine the enzymes. IMPORTANCE Oxygenic photoautotrophic bacteria, namely, cyanobacteria, have the tricarboxylic acid (TCA) cycle. Recently, metabolite production using the cyanobacterial TCA cycle has been well studied. To enhance the production volume of metabolites, understanding the biochemical properties of the cyanobacterial TCA cycle is required. Generally, malate dehydrogenase oxidizes malate in the TCA cycle. However, cyanobacterial malate dehydrogenase shows low activity and does not use NADP+ as a coenzyme, unlike other cyanobacterial TCA cycle enzymes. Our analyses revealed that another malate oxidation enzyme, the malic enzyme, mainly oxidizes malate that originates from the cyanobacterial TCA cycle. These findings provide better insights into metabolite production using the cyanobacterial TCA cycle. Furthermore, our findings suggest that the enzymes related to the TCA cycle vary from organism to organism and emphasize the importance of analyses to identify the enzymes such as those performed in this study.

Dataset Information

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme).

Publications

Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme).

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets