Fluorescent inhibitor probes of enzyme active site conformation: anion binding to angiotensin-converting enzyme |
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| Authors: | M Martin B L Vallee J F Riordan |
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| Institution: | 1. Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts 02115 USA;2. Brigham and Women''s Hospital, 250 Longwood Avenue, Boston, Massachusetts 02115 USA;1. Department of Plant Biology and Genome Center, University of California, Davis, Davis, CA 95616, USA;2. Plant Ecophysiology, Institute of Environmental Biology, Utrecht University, 3584 Utrecht, the Netherlands;3. Plant Biology Graduate Group, University of California, Davis, Davis, CA 95616, USA;4. Integrative Genetics and Genomics Graduate Group, University of California, Davis, Davis, CA 95616, USA;5. Center for Plant Cell Biology, Department of Botany and Plant Sciences, University of California, Riverside, Riverside, CA 92521, USA;6. IBBM, FCE-UNLP CONICET, La Plata 1900, Argentina;7. Department of Cell and Systems Biology/Centre for the Analysis of Genome Evolution and Function, University of Toronto, 25 Willcocks St., Toronto, ON M5S 3B2, Canada;8. Department of Plant Biology, University of California, Davis, Davis, CA 95616, USA;9. Fort Valley State University, Fort Valley, GA 31030, USA;10. Department of Biomedical Engineering and Genome Center, University of California, Davis, Davis, CA 95616, USA;11. Department of Biology, Emory University, Atlanta, GA 30322, USA;12. Department of Biology, University of Padova, Padova, Italy;13. Department of Plant Sciences, University of California, Davis, Davis, CA 95616, USA;14. Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway;1. Institute of Applied Computer Science, Lodz University of Technology, Stefanowskiego 18/22, 90-924 Lodz, Poland;2. Department of Plant Physiology and Biochemistry, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland;1. State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai''an, Shandong, 271018, China;2. College of Agriculture and Bioengineering, Heze University, Heze, Shandong, 274015, China |
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| Abstract: | Dansylated tight-binding inhibitors are effective fluorophoric probes for detecting conformational changes of enzyme active sites. In this study they have been employed to examine the effect of anions on the conformation of angiotensin-converting enzyme. The efficiency of radiationless energy transfer between enzyme tryptophan residues and an active site-bound dansyl inhibitor has been shown to be enhanced by the addition of chloride. Half-maximal fluorescence enhancement occurs at about 2 mM chloride and is the same for both N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-phenylalanine Ki,app = 50 nM (pH 7.5, 300 mM NaCl)] and N-(1-carboxyl-5-dansylamino-pentyl)-glycyl-L-lysine (Ki,app = 5.7 nM). Other activating anions also evoke similar increases in enzyme-inhibitor energy transfer. Fluorescence changes are not due to binding additional inhibitor molecules but rather to an anion-induced change in protein conformation. |
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