Abstract
The Ada protein of Escherichia coli employs a [Zn(S-cys)(4)](2)(-) site to repair deoxyribonucleic acid alkyl phosphotriester lesions. The alkyl group is transferred to a cysteine thiolate in a stoichiometric reaction. We describe a functional model for this chemistry in which a thiolate of [(CH(3))(4)N](2)[Zn(SC(6)H(5))(4)] accepts a methyl group from (CH(3)O)(3)PO. The thiolate salt (CH(3))(4)N(SC(6)H(5)) is also active in methyl transfer, but the thiol C(6)H(5)SH fails to react. Conductivity measurements and kinetic studies demonstrate that [(CH(3))(4)N](2)[Zn(SC(6)H(5))(4)] forms ion pairs in dimethyl sulfoxide (DMSO) solution (K(IP) = 13 +/- 4 M(-)(1)) which exhibit diminished reactivity. The reaction of [Zn(SC(6)H(5))(4)](2)(-) with (CH(3)O)(3)PO is first order with respect to each reagent. A second-order rate constant for this reaction, k(Zn), was determined to be (1.6 +/- 0.3) x 10(-)(2) M(-)(1) s(-)(1). From kinetic data and equilibria studies, all reactivity of [(CH(3))(4)N](2)[Zn(SC(6)H(5))(4)] toward (CH(3)O)(3)PO could be attributed to dissociated thiolate. Metal complexes representing alternative protein sites were prepared and displayed the following kinetic trend of methyl transfer ability: [(CH(3))(4)N](2)[Zn(SC(6)H(5))(4)] > [(CH(3))(4)N](2)[Co(SC(6)H(5))(4)] approximately [(CH(3))(4)N](2)[Cd(SC(6)H(5))(4)] > [(CH(3))(4)N][Zn(SC(6)H(5))(3)(MeIm)] > [Zn(SC(6)H(5))(2)(MeIm)(2)], where MeIm = 1-methylimidazole. These results are consistent with a dissociated thiolate being the active species and suggest that a similar mechanism might apply to alkyl phosphotriester repair by Ada.
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