Kinetic mechanism of the Mg2+-dependent nucleotidyl transfer catalyzed by T4 DNA and RNA ligases.
The Mg(2+)-dependent adenylylation of the T4 DNA and RNA ligases was studied in the absence of a DNA substrate using transient optical absorbance and fluorescence spectroscopy. The concentrations of Mg(2+), ATP, and pyrophosphate were systematically varied, and the results led to the conclusion that the nucleotidyl transfer proceeds according to a two-metal ion mechanism. According to this mechanism, only the di-magnesium-coordinated form Mg(2)ATP(0) reacts with the enzyme forming the covalent complex E.AMP. The reverse reaction (ATP synthesis) occurs between the mono-magnesium-coordinated pyrophosphate form MgP(2)O(7)(2-) and the enzyme.MgAMP complex. The nucleotide binding rate decreases in the sequence ATP(4-) > MgATP(2-) > Mg(2)ATP(0), indicating that the formation of the non-covalent enzyme.nucleotide complex is driven by electrostatic interactions. T4 DNA ligase shows notably higher rates of ATP binding and of subsequent adenylylation compared with RNA ligase, in part because it decreases the K(d) of Mg(2+) for the enzyme-bound Mg(2)ATP(0) more than 10-fold. To elucidate the role of Mg(2+) in the nucleotidyl transfer catalyzed by T4 DNA and RNA ligases, we propose a transition state configuration, in which the catalytic Mg(2+) ion coordinates to both reacting nucleophiles: the lysyl moiety of the enzyme that forms the phosphoramidate bond and the alpha-beta-bridging oxygen of ATP.
Cherepanov
Alexei V
AV
Kluyver Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft 2628 BC, The Netherlands.
de Vries
Simon
S
D000255:Adenosine Triphosphate / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology*; D002384:Catalysis; D011088:DNA Ligases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000255:Adenosine Triphosphate; D011088:DNA Ligases; D011130:RNA Ligase (ATP); D008274:Magnesium