Enzymatic effects of a lysine-to-glutamine mutation in the ATP-binding consensus sequence in the RecD subunit of the RecBCD enzyme from Escherichia coli.
The RecBCD-K177Q enzyme has a lysine-to-glutamine mutation in the putative ATP-binding sequence of the RecD protein (Korangy, F., and Julin, D.A. (1992) J. Biol. Chem. 267, 1727-1732). We have compared the enzymatic properties of the RecBCD-K177Q enzyme with those of the wild-type RecBCD enzyme from Escherichia coli. The purified RecBCD-K177Q enzyme has ATP-dependent nuclease activity on double-stranded or denatured DNA which is reduced (4-14-fold less) compared with the wild type. The kcat and Km(ATP) for ATP hydrolysis stimulated by double-stranded DNA are both reduced in RecBCD-K177Q, so that kcat/Km(ATP) is relatively unaffected. The mutant enzyme is impaired in its ability to unwind DNA in an assay where single-stranded DNA is trapped by the single-stranded DNA binding protein and subsequently degraded by S1 nuclease. The mutant enzyme also produces fewer acid-soluble DNA nucleotides per ATP hydrolyzed than does the wild type, at low ATP concentrations (less than 20 microM).
Korangy
F
F
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
Julin
D A
DA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D004265:DNA Helicases / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D005973:Glutamine; D000255:Adenosine Triphosphate; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D008239:Lysine