Photoaffinity labeling of the recBCD enzyme of Escherichia coli with 8-azidoadenosine 5'-triphosphate.
The recB and recD subunits of the recBCD enzyme (exonuclease V) from Escherichia coli were covalently photolabeled with the ATP photoaffinity analogue [alpha-32P]8-azido-ATP. The labeling was specific for ATP binding sites by the following criteria. Saturation occurs at high 8-azido-ATP concentrations with dissociation constants of 30 and 120 microM for the recD and recB subunits, respectively; ATP strongly inhibits the photolabeling; 8-azido-ATP is hydrolyzed by the recBCD enzyme and supports its double-stranded DNA exonuclease activity; and the label is largely confined to two peptides obtained by tryptic digestion of the photolabeled holoenzyme; one is derived from the recB subunit and the other from the recD subunit.
Julin
D A
DA
Lehman
I R
IR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D046911:Macromolecular Substances; D009711:Nucleotides / Q000494:pharmacology; D010777:Photochemistry; D010957:Plasmids; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D004279:DNA, Viral; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009711:Nucleotides; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D014357:Trypsin; D005634:Fructose-Bisphosphate Aldolase