Fluorine-19 nuclear magnetic resonance and biochemical characterization of fluorotyrosine-labeled-thymidylate-synthetase |
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| Institution: | 1. School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan;2. Department of Beauty Science, National Taichung University of Science and Technology, No.193, Sec.1, San-Min Rd., Taichung City, Taiwan;3. Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan;1. Department of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan;2. Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan;1. Life Science Group, Scientific Research Division, National Synchrotron Radiation Research Center, Hsinchu, Taiwan;2. School of Biomedical Sciences, Chung Shan Medical University, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan;3. Department of Beauty Science, National Taichung University of Science and Technology, No.193, Sec.1, San-Min Rd., Taichung City, Taiwan;4. Department of Biotechnology and Bioindustry Sciences, National Cheng Kung University, Tainan City, Taiwan;5. Department of Physics, National Tsing Hua University, Hsinchu, Taiwan;6. Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan;7. Department of Medical Research, Chung Shan Medical University Hospital, No.110, Sec.1, Chien-Kuo N. Rd., Taichung City, Taiwan |
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| Abstract: | Fluorotyrosine has been incorporated into thymidylate synthetase from Lactobacillus casei by growth of the bacterium in media containing 3-fluorotyrosine. The enzyme exhibited a specific activity 70% of that of the normal enzyme and formed a covalent binary complex with pyrimidine nucleotides, as well as a covalent ternary complex with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate. 19F nuclear magnetic resonance spectroscopy has been used to follow the formation of these complexes. 5-Fluorodeoxyuridylate, dUMP, dTMP and dCMP produced identical conformational changes in the enzyme as monitored by the fluorotyrosyl resonances. Ternary complex formation of the fluorotyrosine-containing enzyme with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate resulted in further spectral changes. |
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