Back to Search
Start Over
Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)
- Source :
- Biochemical Journal. 392:39-45
- Publication Year :
- 2005
- Publisher :
- Portland Press Ltd., 2005.
-
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 Km values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate Km 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 Kact 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.
- Subjects :
- Models, Molecular
Stereochemistry
Molecular Sequence Data
Allosteric regulation
Malic enzyme
Gene Expression
Biology
Biochemistry
Malate dehydrogenase
Substrate Specificity
Enzyme activator
Fumarates
Malate Dehydrogenase
Humans
Amino Acid Sequence
Enzyme kinetics
Molecular Biology
chemistry.chemical_classification
Aspartic Acid
Cell Biology
Fumarate reductase
Recombinant Proteins
Mitochondria
Enzyme Activation
Kinetics
Enzyme
chemistry
Mutation
NAD+ kinase
Allosteric Site
Research Article
Subjects
Details
- ISSN :
- 14708728 and 02646021
- Volume :
- 392
- Database :
- OpenAIRE
- Journal :
- Biochemical Journal
- Accession number :
- edsair.doi.dedup.....cbe346ea68334e4d3ecd159811b61074