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Permanent link (DOI): https://doi.org/10.7939/R3T727V7Z
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Spectroscopic characterization of recombinant dihydrolipoamide dehydrogenase and its site-directed mutants Open Access
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Korotchkina, Lioubov G.
Hyatt, Susannah L.
Kav, Nataraj N. V.
Patel, Mulchand S.
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In this paper, we report the overexpression and single-step purification of recombinant wild-type and site-directed mutants of human dihydrolipoamide dehydrogenase in Escherichia coli and detailed spectroscopic studies aimed at understanding the catalytic mechanism of this enzyme. One mutation (K37E) has been identified in a patient lacking dihydrolipoamide dehydrogenase activity and has been reported previously (Liu, T.-C., Kim, H., Arizmendi, C., Kitano, A., and Patel, M. S.(1993) Proc. Natl. Acad. Sci. USA. 90, 5186-5190), while the other two mutations were previously generated specifically to address the role of the active-site base (His-452) and its ion pair (Glu-457). Circular dichroic and fluorescence spectroscopic data illustrate the role of these amino acids in maintaining the structure and function of human dihydrolipoamide dehydrogenase. While mutant H452Q is severely crippled in catalysis of the physiological reaction, the reverse reaction is affected in the E457Q mutant. The K37E mutant shows very little deviation from the wild-type enzyme.
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- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Liu, T. -C., Korotchkina, L., Hyatt, S. L., Kav, N. N. V., & Patel, M. S. (1995). Spectroscopic characterization of recombinant dihydrolipoamide dehydrogenase and its site-directed mutants. Journal of Biological Chemistry, 270(26), 15545-15550. http://dx.doi.org/10.1074/jbc.270.26.15545
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