Abstract
The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl transfer process is autocatalyzed by coordination of the acceptor residue, Cys69, to a tightly bound zinc ion. Kinetic data reveal a 4-fold reduction in the methylphosphotriester repair activity for the Cd(II) form of Ada versus the native Zn(II)-bound form, thus confirming a direct role for the metal in autocatalysis. Quantitative electrophoretic mobility shift assays reveal that the specific DNA affinity of the protein is increased 10(3)-fold by transfer of a methyl group to Cys69; the Cd(II) and the Zn(II) forms of the protein behave similarly in this respect. This methylation-sensitive stimulation of binding underlies the ability of Ada to activate inducibly the transcription of a methylation-dependent regulon. We conclude that the chemical properties of the bound metal influence the transition state for autocatalytic methyl transfer, but not the structure that ultimately results from this process.
Highlights
From t~e tprogram for Higher Degrees in Biophysics and the Wepartment of Chemistry, Harvard University, Cambridge, Massachusetts 02138
The methyl transfer process is autocatalyzed by coordination of the acceptor residue, Cy s69, to a tightly bound zinc ion
We conclude that the chemical properties of the bound metal influence the transition state for autocatalytic methyl transfer, but not the structure that results from this process
Summary
From t~e tprogram for Higher Degrees in Biophysics and the Wepartment of Chemistry, Harvard University, Cambridge, Massachusetts 02138. Quantitative electrophoretic mobility shift assays reveal that the specific DNA affinity of the protein is increased 103.fold by transfer of a methyl group to Cys; the Cd(II) and the Zn(II) forms of the protein behave in this reo spect This methylation-sensitive stimulation ofbinding underlies the ability of Ada to activate inducibly the transcription of a methylation-dependent regulon. These structural studies identify the N terminus active site cysteine, Cys , as one of the four metal ligand residues in Ada, thereby implicating the metal ion in stabilization of the protein structure, and in direct metalloactivation of the methyl acceptor residue
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