The associative nature of adenylyl transfer catalyzed by T4 DNA ligase

Abstract

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.chemical movie ͉ nucleotidyl transfer ͉ structural reaction kinetics ͉ time-resolved cryo-magic-angle-spinning NMR ͉ transition state U nderstanding chemical mechanics of biocatalysis is a fundamental goal of life sciences. With the development of high-resolution x-ray diffraction analysis and solution NMR a large number of protein structures in the resting state have been solved, giving knowledge on how proteins look, e.g., the detailed view of protein architecture on the primary, secondary, tertiary, and quaternary structure levels. Further insight is coming with studies on how proteins work, e.g., by observing changes of the protein structure in the course of a chemical reaction-recording a molecular movie. Femtosecond laser pulses, molecular beams and ultrafast electron diffraction are used in (in)organic chemistry for monitoring breaking and forming of chemical bonds in real time (1). In biochemistry, kinetic crystallography is used to record molecular movies, an approach that combines starting and stopping the reaction in a protein crystal with x-ray data acquisition at low temperatures (2-8). The successful outcome of a time-resolved x-ray experiment depends as much on a prompt triggering of the reaction as on preparing well diffracting protein crystals. Here we introduce time-resolved lowtemperature magic-angle-spinning (cryo-MAS) NMR spectroscopy as a complementary ''noninvasive'' technique to study catalytic dynamics of biochemical reactions. It combines phototriggering and freeze-trapping with real-time monitoring of chemical transformations and requires neither protein crystallization nor high intensity penetrating radiation beams. We have assayed the nucleotidyl transfer reaction catalyzed by DNA ligase from bacteriophage T4, a Mg 2ϩ -and AT...

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