The Associative Nature of Adenylyl Transfer Catalyzed by T4 DNA Ligase

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD⁺ and assistance of a divalent metal cofactor Mg²⁺. Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell r...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2008-06, Vol.105 (25), p.8563-8568
Main Authors: Cherepanov, Alexey V., Doroshenko, Elena V., Matysik, Jörg, de Vries, Simon, de Groot, Huub J. M.
Format: Article
Language:English
Subjects:
Adenosine triphosphatase
Adenosine Triphosphate - chemistry
Bacteriophage T4 - enzymology
Biochemistry
Biological Sciences
Catalysis
Cells
Chemical bonding
Deoxyribonucleic acid
Diphosphates
DNA
DNA Ligases - chemistry
DNA Ligases - metabolism
Enzymes
Heparin
Kinetics
Magnesium - chemistry
Magnesium - metabolism
Magnetic Resonance Spectroscopy
NAD - chemistry
NAD - metabolism
Phosphates
Phosphoranes
Reaction kinetics
Resonance
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Summary:DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD⁺ and assistance of a divalent metal cofactor Mg²⁺. 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 ³¹P 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 Mg²⁺, 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0709140105