Alkyl transfer to metal thiolates: Kinetics, active species identification, and relevance to the DNA methyl phosphotriester repair center of Escherichia coli Ada

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 [(CH3)(4)N](2)[Zn(SC6H5)(4)] accepts a methyl group from (CH3O)(3)PO. The thiolate salt (CH3)(4)N(SC6H5) is also active in methyl transfer, but the thiol C6H5SH fails to-react. Conductivity measurements and kinetic studies demonstrate that [(CH3)(4)N](2)[Zn(SC6H5)(4)] forms ion pairs in dimethyl sulfoxide (DMSO) solution (K-IP = 13 +/- 4 M(-1)) which exhibit diminished reactivity. The reaction of [Zn(SC6H5)(4)](2-) With (CH3O)(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 [(CH3)(4)N](2)[Zn(SC6H5)(4)] toward (CH3O)(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: [(CH3)(4)N](2)[Zn(SC6H5)(4)] > [(CH3)(4)N](2)[CO(SC6H5)(4)] approximate to [(CH3)(4)N](2)[Cd(SC6H5)(4)] > [(CH3)(4)N][Zn(SC6H5)(3)(MeIm)] > [Zn(SC6H5)(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.