Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA.
T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
N
N
Hori
T
T
Doi
Y
Y
Karaki
M
M
Kikuchi
M
M
Ikehara
E
E
Ohtsuka
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins
D005971:Glutamates; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D018698:Glutamic Acid; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)