Alteration by site-directed mutagenesis of the conserved lysine residue in the ATP-binding consensus sequence of the RecD subunit of the Escherichia coli RecBCD enzyme.
The RecD subunit of the RecBCD enzyme from Escherichia coli contains an amino acid sequence common to many enzymes which bind ATP or GTP (Gly-X-X-Gly-X-Gly-Lys-Thr). We have changed the conserved lysine residue (amino acid number 177) in the RecD protein to glutamine to investigate the role of RecD, and ATP-binding to RecD, in the enzymatic activities of RecBCD. The mutant RecD protein assembles with the RecB and RecC subunits and the mutant enzyme, designated RecBCD-K177Q, can be purified in the same way as the wild-type RecBCD enzyme. The mutant RecD subunit in RecBCD-K177Q is photolabeled to a lesser extent by the ATP analogue 8-azido-adenosine-5'-triphosphate than is the wild-type RecD subunit in RecBCD, suggesting that the mutation has reduced the affinity of RecD for ATP.
Korangy
F
F
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Julin
D A
DA
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D000345:Affinity Labels; D001386:Azides; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D008239:Lysine