| Autor: |
Alexey V. Cherepanov, Huub J. M. de Groot, Simon de Vries, Jörg Matysik, Elena V. Doroshenko |
| Jazyk: |
angličtina |
| Rok vydání: |
2008 |
| Předmět: |
|
| Zdroj: |
Proceedings of the National Academy of Sciences, 105(25), 8563-8568 |
| Popis: |
DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD + and assistance of a divalent metal cofactor Mg 2+ . Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover 31 P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase–nucleotide complex is triggered in situ by the photo release of caged Mg 2+ , and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4–5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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