Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis.
Vaccinia DNA topoisomerase, a 314-amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. To identify amino acids that participate in the transesterification reaction, we introduced alanine substitutions at 39 positions within a conserved 57amino acid segment upstream of the active-site tyrosine. Purified wild type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA. The majority of mutant proteins displayed wild type topoisomerase activity. Mutant enzymes that relaxed DNA at reduced rates were subjected to kinetic analysis of the strand cleavage and religation steps under single-turnover and equilibrium conditions. For the wild type topoisomerase, the observed single-turnover cleavage rate constant (kcl) was 0.29 s-1 and the cleavage-religation equilibrium constant (Kcl) was 0.22. The most dramatic mutational effects were seen with R223A; removal of the basic side chain reduced the rates of cleavage and religation by factors of 10(-4.3) and 10(-5.0), respectively, and shifted the cleavage-religation equilibrium in favor of the covalently bound state (Kcl = 1). Introduction of lysine at position 223 restored the rate of cleavage to 1/10 that of the wild type enzyme. We conclude that a basic residue is essential for covalent catalysis and suggest that Arg-223 is a constituent of the active site. Modest mutational effects were observed at two other positions (Lys-220 and Asn-228), at which alanine substitutions slowed the rates of strand cleavage by 1 order of magnitude and shifted the equilibrium toward the noncovalently bound state. Arg-223 and Lys-220 are conserved in all members of the eukaryotic type I topoisomerase family; Asn-228 is conserved among the poxvirus enzymes.
Cheng
C
C
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Wang
L K
LK
Sekiguchi
J
J
Shuman
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D001120:Arginine / Q000032:analysis; D001224:Aspartic Acid / Q000032:analysis; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics*; D004278:DNA, Superhelical / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000032:analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D014616:Vaccinia virus / Q000201:enzymology*
D000596:Amino Acids; D004278:DNA, Superhelical; D001224:Aspartic Acid; D001120:Arginine; D004264:DNA Topoisomerases, Type I; D008239:Lysine