Interaction of pyridoxal phosphate with thymidylate synthase: Spectral and equilibrium dialysis studies |
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| Affiliation: | 1. Osakidetza Basque Health Service, Debagoiena Integrated Health Organisation, Pharmacy Service, Nafarroa Hiribidea, 16, 20500 Arrasate, Gipuzkoa, Spain;2. Biocruces Bizkaia Health Research Institute, ES48903 Barakaldo, Biscay, Spain;3. Department of Analytical Chemistry, University of the Basque Country, ES48940 Leioa, Biscay, Spain;4. Research Centre for Experimental Marine Biology & Biotechnology, ES48620 Plentzia, Biscay, Spain;5. Department of Analytical Chemistry, Faculty of Science and Technology, University of the Basque Country, Bilbao, Spain;6. Research Centre for Experimental Marine Biology and Biotechnology, University of the Basque Country (PiE-UPV/EHU), Plentzia, Basque Country 48620, Spain;7. Pharmacy Service, Araba-Integrated Health Care Organization, Santiago Hospital, Vitoria-Gasteiz, Alava, Spain;8. Pharmacy Service, Araba Integrated Health Care Organization, Txagorritxu Hospital, Vitoria-Gasteiz, Alava, Spain;9. NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country UPV/EHU, Paseo de la Universidad 7, Vitoria-Gasteiz 01006, Spain;10. Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain;11. University Institute for Regenerative Medicine and Oral Implantology - UIRMI (UPV/EHU-Fundación Eduardo Anitua), Vitoria, Spain;12. Singapore Eye Research Institute, The Academia, 20 College Road, Discovery Tower, Singapore;13. Bioaraba, NanoBioCel Research Group, Vitoria-Gasteiz, Spain;14. Bioaraba Health Research Institute, Osakidetza Basque Health Service, Araba Mental Health Network, Araba Psychiatric Hospital, Pharmacy Service, c/Alava 43, 01006 Vitoria-Gasteiz, Alava, Spain |
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| Abstract: | - 1.1. Changes in the spectrum of pyridoxal phosphate (PLP) were produced by adding an equimolar amount of native thymidylate synthase, but not by adding denatured enzyme or enzyme modified by sulfhydryl-blocking reagents.
- 2.2. The dissociation constant of the thymidylate synthase-PLP complex determined by equilibrium dialysis was 9 ± 1.6 μM, the maximum number of PLP molecules bound per molecule of native thymidylate synthase was 2.5 ± 0.4, and the Hill coefficient was 0.97.
- 3.3. No evidence of PLP binding was found with denatured thymidylate synthase, and only slight binding was observed when enzyme SH groups were blocked or when the active site was blocked with 5-fluorodeoxyuridylate (FdUMP) and methylenetetrahydrofoliate.
- 4.4. The presence of dUMP, dTMP, or FdUMP interfered with the binding of PLP to thymidylate synthase, and the presence of equimolar amounts of PLP interfered with the binding of dUMP.
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