Mechanistic Studies of the Inhibition of MutT dGTPase by the Carcinogenic Metal Ni(II)

Abstract

Promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) levels are increased in DNA of animals exposed to carcinogenic metals, such as Ni(II). Besides being generated directly in genomic DNA, 8-oxo-dG may be incorporated there from 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), a product of oxidative damage to the nucleotide pool. The Escherichia coli dGTPase MutT, and analogous dGTPases in rats and humans, have been suggested as a defense against such incorporation because they hydrolyze 8-oxo-dGTP to 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP). MutT and its mammalian counterparts are Mg(II)-dependent enzymes. Ni(II), in turn, is known to interact antagonistically with Mg(II) in biological systems. Thus, we hypothesized that Ni(II) might inhibit the activity of MutT. As an initial examination of this hypothesis, we conducted enzyme kinetic studies of MutT to determine the effect of Ni(II) on MutT activity and the mechanisms involved. As found, Ni(II) inhibited MutT in a concentration-dependent manner when either dGTP or 8-oxo-dGTP was the nucleotide substrate. Ni(II) was determined to be an uncompetitive inhibitor of MutT with respect to Mg(II) when dGTP was the substrate, with apparent Ki of 1.2 mM Ni(II), and a noncompetitive inhibitor with respect to Mg(II) when 8-oxo-dGTP was the substrate, with apparent Ki of 0.9 mM Ni(II). Hence, the two metal cations did not compete with each other for binding at the MutT active site. This makes it difficult to predict Ni(II) effects on 8-oxo-dGTPases of other species. However, based upon the amino acid sequences of human and rat MutT-like dGTPases, their capacity for Ni(II) binding should be greater than that of MutT. Whether this could lead to stronger inhibition of those enzymes by Ni(II), or not, remains to be investigated.

Metal Complexes as Enzyme Inhibitors

“…191 Ni-(II) has been observed to inhibit MutT-dGTPase, which is involved in the repair of 8-oxo-dGTP lesions in DNA (millimolar K i ). 192 Cells under oxidative stress form 8-oxo-dGTP, and the presence of this nucleotide increases the mutation frequency of A to C transversions due to 8-oxo-dG-dA mispairing. Inhibition of the Mg(II)-dependent MutT repair enzyme can lead to increased mutagenesis.…”

“…Modeling studies suggest that Ni(II) does not appear to bind to the Mg(II) binding site but induces conformational changes which are predicted to occur through binding to histidine residues. 192 Zinc and cadmium have also been seen to inhibit DNA repair enzymes (see review; 191 note entire issue devoted to mechanisms of metal genotoxicity). Purified DNA ligase is inhibited by 0.8 mM ZnCl 2 and 0.04 mM CdCl 2 .…”

“…Inhibition of DNA replication and repair has been strongly implicated in the mutagenicity and carcinogenicity of metal ions . Ni(II) has been observed to inhibit MutT-dGTPase, which is involved in the repair of 8-oxo-dGTP lesions in DNA (millimolar K i ) . Cells under oxidative stress form 8-oxo-dGTP, and the presence of this nucleotide increases the mutation frequency of A to C transversions due to 8-oxo-dG-dA mispairing.…”

“…Inhibition of the Mg(II)-dependent MutT repair enzyme can lead to increased mutagenesis. Modeling studies suggest that Ni(II) does not appear to bind to the Mg(II) binding site but induces conformational changes which are predicted to occur through binding to histidine residues …”

Structure of a Nudix protein fromPyrobaculum aerophilumreveals a dimer with two intersubunit β-sheets

“…Their functions are mainly to reduce the level of these potentially toxic compounds and the accumulation of metabolic intermediates (Safrany et al, 1998;O'Handley et al, 2001). The best-studied Nudix enzyme is the MutT protein from Escherichia coli (Shimokawa et al, 2000;Abeygunawardana et al, 1995;Taddei et al, 1997;Wagner et al, 1997;Bhatnagar et al, 1991;Frick et al, 1995;Porter et al, 1996). It reduces the rate of AT to CG transversion several thousand-fold by hydrolyzing the mutagenic nucleotide 8-oxo-dGTP to give 8-oxo-dGMP, thus preventing it from being incorporated into DNA (Bessman et al, 1996).…”