Catalytic mechanism and DNA substrate recognition of Escherichia coli MutY protein.
Escherichia coli MutY protein cleaves A/G- or a/7,8-dihydro-8-oxo-guanine (A/GO)-containing DNA on the A-strand by N-glycosylase and apurinic/apyrimidinic endonuclease or lyase activities. In this paper, we show that MutY can be trapped in a stable covalent enzyme-DNA intermediate in the presence of sodium borohydride, a new finding that supports the grouping of MutY in that class of DNA glycosylases that possess concomitant apurinic/apyrimidinic lyase activity. To potentially help determine the substrate recognition site of MutY, mutant proteins were constructed. MutY proteins with a Gly116 --> Ala (G116A) or Asp (G116D) mutation had reduced binding affinities for both A/G- and A/GO-containing DNA substrates. The catalytic parameters, however, were differentially affected. While A/G- and A/GO-containing DNA were cleaved by MutY with specificity constants (kcat/Km) of 10 and 3.3 min-1 microM-1, respectively, MutY(G116D) cleaved these DNAs 2, 300- and 9-fold less efficiently. The catalytic activities of MutY(G116A) with A/G- and A/GO-containing DNA were about the same as that of wild-type MutY. Both MutY(G116A) and MutY(G116D) could be trapped in covalent intermediates with A/GO-containing DNA, but with lower efficiencies than the wild-type enzyme in the presence of sodium borohydride. MutY(G116A) also formed a covalent intermediate with A/G-containing DNA, but MutY(G116D) did not. Since Gly116 of MutY lies in a region that is highly conserved among several DNA glycosylases, it is likely this conserved region is in the proximity of the substrate binding and/or catalytic sites.
Lu
A L
AL
Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, Maryland 21201, USA.
Yuen
D S
DS
Cillo
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D045647:DNA Glycosylases; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase