Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine.
We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP-ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 microM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the 4-formyl group of the pyridoxyl moiety of PLP-AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/mol of gyrase. The Km for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition. A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys-110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.
Tamura
J K
JK
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Gellert
M
M
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives*; D012689:Sequence Homology, Nucleic Acid
D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine