Catalytic mechanism of DNA topoisomerase IB.
Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 5'-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pKa of the 5'-S leaving group versus 5'-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.
Krogh
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
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D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D014443:Tyrosine / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D014764:Viral Proteins; D014443:Tyrosine; D006639:Histidine; D004247:DNA; D001120:Arginine; D004264:DNA Topoisomerases, Type I; D008239:Lysine