Effect of Mg(II) Binding on the Structure and Activity ofEscherichia coli DNA Topoisomerase I

Abstract

Escherichia coli DNA topoisomerase I is the best characterized bacterial type I DNA topoisomerases (for review, see Refs. 1 and 2). Its major function in vivo is the removal of negative supercoils from DNA (3, 4). In vitro, such relaxation activity requires that Mg(II) be present in the reaction mixture (3, 5).When other divalent ions were tested (5), Ca(II) could partly replace the Mg(II) in the relaxation of negatively supercoiled DNA while the presence of other divalent ions, such as Mn(II), did not support the relaxation of negatively supercoiled DNA by the enzyme. Moreover, the presence of 2 mM Mn(II) had an inhibitory effect when co-incubated with the enzyme and 2 mM Mg(II) (5). These results suggested that there are specific interactions between Mg(II) and the enzyme or enzyme⅐DNA complex.The three-dimensional structure of the 67-kDa N-terminal fragment of E. coli DNA topoisomerase I has been determined by x-ray crystallography (6). It was noted (6) that near the active site nucleophile Tyr-319, there are three acidic residues, Asp-111, Asp-113, and Glu-115 arranged similarly to the three acidic residues known to coordinate two divalent ions in Klenow fragment (7). According to the models proposed for DNA polymerase mechanism, these coordinated divalent ions are essential for the nucleotidyl transfer catalytic activity (8, 9) of DNA polymerases. However, divalent ions were not present in the topoisomerase I crystal structure (6). It is also known that Mg(II) is not required for DNA cleavage by topoisomerase and formation of the covalent protein-DNA intermediate (10) although Mg(II) is required for intermolecular religation to be observed (11). It remains unclear if Mg(II) interacts directly with the enzyme.The basis for the requirement of Mg(II) for relaxation activity needs to be elucidated to fully understand the enzyme mechanism. There are several possible roles for Mg(II) in the relaxation of negatively supercoiled DNA by E. coli DNA topoisomerase I. For a direct role in catalysis, one or more Mg(II) bound at the active site may activate the Tyr-319 hydroxyl nucleophile and stabilize the DNA 3ЈOH-leaving group during the DNA strand cleavage step, and/or they may activate the DNA 3ЈOH as the attacking nucleophile and stabilze the Tyr-319 hydroxyl-leaving group during the DNA religation step. More indirectly, Mg(II) coordination may place the DNA phosphates and enzyme catalytic groups in the positions required for catalysis. In addition, binding of Mg(II) may allow the enzyme to undergo the conformational changes postulated to be required for the strand passage, DNA religation and substrate release steps in the proposed mechanism of reaction (6).The fluorescence of tryptophan residues is highly dependent on their local environment and enzyme conformation. We demonstrated here that significant changes in enzyme structure could be observed in the presence of 2 mM MgCl 2 , affecting the tryptophan emission intensity. This suggests that Mg(II) binding may allow the enzyme to assume a conformation necessary ...

The Acidic Triad Conserved in Type IA DNA Topoisomerases Is Required for Binding of Mg(II) and Subsequent Conformational Change

“…Double and triple mutants lacking two or more of these acidic residues have now been purified for analysis of their enzymatic activities. The results presented here showed that relaxation activity for double mutants involving Asp-111, Asp-113, and Glu-115 requires higher concentrations of Mg(II) for maximal activity, supporting the hypothesis that the carboxylates in these three conserved acidic residues are involved in coordinating at least one of the two Mg(II) required for relaxation activity (13). We have also developed a proteolytic digestion assay using Glu-C endoproteinase to detect the Mg(II)-induced conformational change in E. coli DNA topoisomerase I.…”

“…We have previously shown that following incubation of the wild type topoisomerase I with 2 mM MgCl 2 , around 2 Mg(II) remains bound to each enzyme molecule after dialysis (13). Similar treatment of the acidic triad double and triple mutants followed by inductively coupled plasma analysis of the Mg(II) content (Table I) showed that their Mg(II) binding stoichiometries were reduced, in correlation with the relaxation activities observed.…”

“…With wild type enzyme, such high concentration of MgCl 2 resulted in inhibition of relaxation activity. Previous studies showed that as little as 1.5-2.5 mM was sufficient for maximal activity for the wild type topoisomerase I (13,18). Relaxation activity could not be restored for the D111A/D113A/E115A triple mutant even at 20 mM MgCl 2 , and only partial relaxation of the input DNA can be achieved with the D111A/D113A mutant.…”

“…We have previously shown that Mg(II) binding result in a conformational change in topoisomerase I detectable by decreased tryptophan fluorescence (13). Analysis of the fluorescence data (Fig.…”

Effect of Mg2+ on the thermal inactivation and unfolding of creatine kinase

“…ATP±Mg) [6,7]. The third mechanism is through allosteric activation in which binding to one or www.elsevier.com/locate/ijbcb more forms of the enzyme in the reaction course induces a change in its conformation [8,9].…”