Histidine 265 is important for covalent catalysis by vaccinia topoisomerase and is conserved in all eukaryotic type I enzymes.
Vaccinia topoisomerase catalyzes DNA cleavage and rejoining via transesterification to pentapyrimidine recognition site 5'-(C/T)CCTT downward arrow in duplex DNA. The proposed reaction mechanism involves general-base catalysis of the attack by active site nucleophile Tyr-274 on the scissile phosphodiester and general-acid catalysis of the expulsion of the 5'-deoxyribose oxygen on the leaving DNA strand. The pKa values suggest histidine and cysteine side chains as candidates for the roles of proton acceptor and donor, respectively. To test this, we replaced each of the eight histidines and two cysteines of the vaccinia topoisomerase with alanine. Single mutants C100A and C211A and a double mutant C100A-C211A were fully active in DNA relaxation, indicating that a cysteine is not the general acid. Only one histidine mutation, H265A, affected enzyme activity. The rates of DNA relaxation, single-turnover strand cleavage, and single-turnover religation by H265A were 2 orders of magnitude lower than the wild-type rates. Yet the H265A mutation did not alter the dependence of the cleavage rate on pH, indicating that His-265 is not the general base. Replacing His-265 with glutamine or asparagine slowed DNA relaxation and single-turnover cleavage to about one-third of the wild-type rate. All three mutations, H265A, H265N, and H265Q, skewed the cleavage-religation equilibrium in favor of the covalently bound state. His-265 is strictly conserved in every member of the eukaryotic type I topoisomerase family.
Petersen
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
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D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D014616:Vaccinia virus / Q000201:enzymology*
D005973:Glutamine; D006639:Histidine; D001216:Asparagine; D004247:DNA; D004264:DNA Topoisomerases, Type I