Binding of nucleotides by T4 DNA ligase and T4 RNA ligase: optical absorbance and fluorescence studies.
The interaction of nucleotides with T4 DNA and RNA ligases has been characterized using ultraviolet visible (UV-VIS) absorbance and fluorescence spectroscopy. Both enzymes bind nucleotides with the K(d) between 0.1 and 20 microM. Nucleotide binding results in a decrease of absorbance at 260 nm due to pi-stacking with an aromatic residue, possibly phenylalanine, and causes red-shifting of the absorbance maximum due to hydrogen bonding with the exocyclic amino group. T4 DNA ligase is shown to have, besides the catalytic ATP binding site, another noncovalent nucleotide binding site. ATP bound there alters the pi-stacking of the nucleotide in the catalytic site, increasing its optical extinction. The K(d) for the noncovalent site is approximately 1000-fold higher than for the catalytic site. Nucleotides quench the protein fluorescence showing that a tryptophan residue is located in the active site of the ligase. The decrease of absorbance around 298 nm suggests that the hydrogen bonding interactions of this tryptophan residue are weakened in the ligase-nucleotide complex. The excitation/emission properties of T4 RNA ligase indicate that its ATP binding pocket is in contact with solvent, which is excluded upon binding of the nucleotide. Overall, the spectroscopic analysis reveals important similarities between T4 ligases and related nucleotidyltransferases, despite the low sequence similarity.
Cherepanov
A V
AV
Kluyver Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands.
de Vries
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism*; D011088:DNA Ligases / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
D012313:RNA; D014364:Tryptophan; D000255:Adenosine Triphosphate; D004247:DNA; D011088:DNA Ligases