Location of the active site for enzyme-adenylate formation in DNA ligases.
The enzyme-AMP reaction intermediate of the 102-kDa bovine DNA ligase I was digested with trypsin, and the adenylylated peptide was isolated by chromatography under conditions that maintain the acid-labile phosphoramidate bond. Microsequencing of the peptide showed that it contains an internal trypsin-resistant lysine residue, as expected for the site of adenylylation. Inhibition of DNA ligase I activity by pyridoxal 5'-phosphate also indicated the presence of a reactive lysine residue in the catalytic domain of the enzyme. Comparison of the known primary structures of several other DNA ligases with the adenylylated region of mammalian DNA ligase I allows their active sites to be tentatively assigned by sequence homology. The ATP-dependent DNA ligases of mammalian cells, fission yeast, budding yeast, vaccinia virus, and bacteriophages T3, T4, and T7 contain the active site motif Lys-Tyr/Ala-Asp-Gly-(Xaa)-Arg, with the reactive lysine residue flanked by hydrophobic amino acids. The distance between the postulated adenylylation site and the carboxyl terminus of the polypeptide is very similar in these ATP-dependent DNA ligases, whereas the size of the amino-terminal region is highly variable.
Tomkinson
A E
AE
Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, United Kingdom.
Totty
N F
NF
Ginsburg
M
M
Lindahl
T
T
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D013950:Thymus Gland / Q000201:enzymology; D014357:Trypsin
D010446:Peptide Fragments; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D014357:Trypsin; D011088:DNA Ligases