The associative nature of adenylyl transfer catalyzed by T4 DNA ligase

摘要: DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond ATP or NAD+ and assistance a divalent metal cofactor Mg2+. Molecular details catalysis are essential for understanding mechanism metal-promoted phosphoryl transfer reactions living cell responsible wide range processes, e.g., replication transcription, signaling differentiation, coupling metabolism. Here we report single-turnover 31P solid-state NMR study adenylyl catalyzed by from bacteriophage T4. Formation high-energy covalent ligase–nucleotide complex is triggered situ photo release caged Mg2+, sequentially formed intermediates monitored NMR. Analyses reaction kinetics chemical-shift changes indicate that pentacoordinated phosphorane intermediate builds up to 35% total reacting species after 4–5 h reaction. This direct experimental evidence associative nature ligase. spectroscopy rotating solids introduced as an analytical tool recording molecular movies processes. Presented work pioneers promising direction structural studies biochemical transformations.