Journal of Biomedical Science

, Volume 15, Issue 1, pp 37–46

The role of amino acids T148 and R281 in human dihydrolipoamide dehydrogenase

  • Yi-Chun Wang
  • Shih-Tsung Wang
  • Chuan Li
  • Ling-Yun Chen
  • Wen-Hu Liu
  • Pei-Ru Chen
  • Ming-Chih Chou
  • Ted-Chung Liu
Article

DOI: 10.1007/s11373-007-9208-9

Cite this article as:
Wang, YC., Wang, ST., Li, C. et al. J Biomed Sci (2008) 15: 37. doi:10.1007/s11373-007-9208-9

Abstract

Human dihydrolipoamide dehydrogenase (hE3) is a common component of α-ketoacid dehydrogenase complexes. Mutations of this homodimeric protein cause E3 deficiency and are always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other 17 eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, two amino acids, T148 and R281, were subjected to mutagenesis and four hE3 mutants, T148G, T148S, R281N, and R281K, were expressed and assayed. The specific activities of T148G, T148S, R281N, and R281K are 76.34%, 88.62%, 12.50%, and 11.93% to that of wild-type E3, respectively. The FAD content analysis indicated that the FAD content of these mutant E3s were about 71.0%, 92%, 96%, and 93% that of wild-type E3, respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic data demonstrated that the Kcat of forward reaction of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and R281 play a role in the catalytic function of the E3.

Keywords

dihydrolipoamide dehydrogenase E3 enzyme kinetic assay 

Copyright information

© National Science Council Taipei 2007

Authors and Affiliations

  • Yi-Chun Wang
    • 1
    • 2
  • Shih-Tsung Wang
    • 3
  • Chuan Li
    • 2
  • Ling-Yun Chen
    • 4
  • Wen-Hu Liu
    • 5
  • Pei-Ru Chen
    • 6
  • Ming-Chih Chou
    • 7
    • 8
  • Ted-Chung Liu
    • 3
  1. 1.Institute of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC
  2. 2.Department of Biomedical SciencesChung Shan Medical UniversityTaichungTaiwan, ROC
  3. 3.Department of Nutritional ScienceChung Shan Medical UniversityTaichungTaiwan, ROC
  4. 4.Department of BiochemistryChung Shan Medical UniversityTaichungTaiwan, ROC
  5. 5.Department of Radiation Safety RoomChung Shan Medical UniversityTaichungTaiwan, ROC
  6. 6.Department of Instrument CenterChung Shan Medical UniversityTaichungTaiwan, ROC
  7. 7.Institute of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC
  8. 8.School of MedicineChung Shan Medical UniversityTaichungTaiwan, ROC