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 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.
Biophysical Organic Chemistry/Solid-State NMR, Leiden Institute of Chemistry, Faculty of Mathematics and Natural Sciences, Leiden University, Einsteinweg 55, 2333CC Leiden, The Netherlands. alexey.cherepanov@gmail.com
Alexey V
AV
Cherepanov
Elena V
EV
Doroshenko
Jörg
J
Matysik
Simon
S
de Vries
Huub J M
HJ
de Groot
D000255:Adenosine Triphosphate / Q000737:chemistry*; D017122:Bacteriophage T4 / Q000201:enzymology*; D002384:Catalysis; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000737:chemistry / Q000378:metabolism
D009243:NAD; D000255:Adenosine Triphosphate; D011088:DNA Ligases; D008274:Magnesium