LOCATION OF THE ACTIVE-SITE FOR ENZYME-ADENYLATE FORMATION IN DNA LIGASES

被引:131
作者
TOMKINSON, AE [1 ]
TOTTY, NF [1 ]
GINSBURG, M [1 ]
LINDAHL, T [1 ]
机构
[1] UCL, MIDDLESEX HOSP, LUDWIG INST CANC RES, LONDON W1P 8BT, ENGLAND
关键词
DNA REPLICATION; LYSINE-ADENYLATE; PYRIDOXAL 5'-PHOSPHATE; EVOLUTIONARILY CONSERVED ENZYME;
D O I
10.1073/pnas.88.2.400
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:400 / 404
页数:5
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