Mechanistic studies on E. coli DNA topoisomerase I: divalent ion effects.
E. coli DNA topoisomerase I catalyzes the hydrolysis of short, single stranded oligodeoxynucleotides. It also forms a covalent protein-DNA complex with negatively supercoiled DNA in the absence of Mg2+ but requires Mg2+ for the relaxation of negatively supercoiled DNA. In this paper we investigate the effects of various divalent metals on catalysis. For the relaxation reaction, maximum enzyme activity plateaus after 2.5 mM Mg2+. However, the rate of cleavage of short oligodeoxynucleotide increased linearly between 0 and 15 mM Mg2+. In the oligodeoxynucleotide cleavage reaction, Ca2+, Mn2+, Co2+, and Zn2+ inhibit enzymatic activity. When these metals are coincubated with Mg2+ at equimolar concentrations, the normal effect of Mg2+ is not detectable. Of these metals, only Ca2+ can be substituted for Mg2+ as a metal cofactor in the relaxation reaction. And when Mg2+ is coincubated with Mn2+, Co2+, or Zn2+ at equimolar concentrations, the normal effect of Mg2+ on relaxation is not detectable. We propose that Mg2+ allows the protein-DNA complex to assume a conformation necessary for strand passage and enhance the rate of enzyme turnover.
Domanico
P L
PL
Department of Enzymology, Glaxo Research Laboratories, Research Triangle Park, North Carolina.
Tse-Dinh
Y C
YC
D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D003035:Cobalt / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D004278:DNA, Superhelical; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D003035:Cobalt; D008345:Manganese; D004264:DNA Topoisomerases, Type I; D008274:Magnesium; D015032:Zinc; D002118:Calcium