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

  • Published:
Journal of Biomedical Science

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.

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Authors and Affiliations

  1. Institute of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan, ROC

    Yi-Chun Wang

  2. Department of Biomedical Sciences, Chung Shan Medical University, Taichung, 402, Taiwan, ROC

    Yi-Chun Wang & Chuan Li

  3. Department of Nutritional Science, Chung Shan Medical University, No.110, Sec. 1, Chien-Kuo N. Rd, Taichung, 402, Taiwan, ROC

    Shih-Tsung Wang & Ted-Chung Liu

  4. Department of Biochemistry, Chung Shan Medical University, Taichung, 402, Taiwan, ROC

    Ling-Yun Chen

  5. Department of Radiation Safety Room, Chung Shan Medical University, Taichung, 402, Taiwan, ROC

    Wen-Hu Liu

  6. Department of Instrument Center, Chung Shan Medical University, Taichung, 402, Taiwan, ROC

    Pei-Ru Chen

  7. Institute of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan, ROC

    Ming-Chih Chou

  8. School of Medicine, Chung Shan Medical University, Taichung, 402, Taiwan, ROC

    Ming-Chih Chou

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  1. Yi-Chun Wang
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  8. Ted-Chung Liu
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Correspondence to Ted-Chung Liu.

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Wang, YC., Wang, ST., Li, C. et al. The role of amino acids T148 and R281 in human dihydrolipoamide dehydrogenase. J Biomed Sci 15, 37–46 (2008). https://doi.org/10.1007/s11373-007-9208-9

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  • DOI: https://doi.org/10.1007/s11373-007-9208-9

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