Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/38102
標題: Characterization of the functional role of allosteric site residue Asp(102) in the regulatory mechanism of human mitochondrial NAD(P)(+)-dependent malate dehydrogenase (malic enzyme)
作者: Hung, H.C.
洪慧芝
Kuo, M.W.
Chang, G.G.
Liu, G.Y.
關鍵字: allosteric activation;fumarate;malate dehydrogenase;malic enzyme;mutagenesis;enzyme regulation;ascaris-suum;oxidative decarboxylases;catalytic mechanism;crystal-structure;adrenal-cortex;rat-liver;nad;sequence;fumarate;purification
Project: Biochemical Journal
期刊/報告no:: Biochemical Journal, Volume 392, Page(s) 39-45.
摘要: 
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 Arg(67) and Arg(91). 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 Asp(102), 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, Asp(102) was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp(102) for activation by fumarate. Mutation of Asp(102) 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. values for malate, Mg2+ and NAD(+), but it did not notably elevate k(cat). 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 Asp(102) mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp(102) leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.
URI: http://hdl.handle.net/11455/38102
ISSN: 0264-6021
DOI: 10.1042/bj20050641
Appears in Collections:生命科學系所

Show full item record
 

Google ScholarTM

Check

Altmetric

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.