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.
Skip Nav Destination
Article navigation
November 2005
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
November 08 2005
Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)
Hui-Chih Hung;
Hui-Chih Hung
1
*Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan, Republic of China
1To whom correspondence should be addressed (email hchung@dragon.nchu.edu.tw or email liugy@csmu.edu.tw).
Search for other works by this author on:
Meng-Wei Kuo;
Meng-Wei Kuo
*Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan, Republic of China
Search for other works by this author on:
Gu-Gang Chang;
Gu-Gang Chang
†Faculty of Life Sciences, Institute of Biochemistry, and Structural Biology Program, National Yang-Ming University, Taipei 112, Taiwan, Republic of China
Search for other works by this author on:
Guang-Yaw Liu
Guang-Yaw Liu
1
‡Institute of Immunology, Chung-Shan Medical University, Taichung 402, Taiwan, Republic of China
1To whom correspondence should be addressed (email hchung@dragon.nchu.edu.tw or email liugy@csmu.edu.tw).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
April 20 2005
Revision Received:
June 20 2005
Accepted:
June 30 2005
Accepted Manuscript online:
June 30 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 392 (1): 39–45.
Article history
Received:
April 20 2005
Revision Received:
June 20 2005
Accepted:
June 30 2005
Accepted Manuscript online:
June 30 2005
Citation
Hui-Chih Hung, Meng-Wei Kuo, Gu-Gang Chang, Guang-Yaw Liu; Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme). Biochem J 15 November 2005; 392 (1): 39–45. doi: https://doi.org/10.1042/BJ20050641
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.