The Cys292-->Ala substitution in protein R1 of class I ribonucleotide reductase from Escherichia coli has a global effect on nucleotide binding at the specificity-determining allosteric site.

Ribonucleotide reductase from aerobically grown Escherichia coli is allosterically regulated, both with respect to general activity and substrate specificity. Protein R1, the homodimeric enzyme component which harbours binding sites for allosteric effectors (nucleoside triphosphates) as well as substrates (ribonucleoside diphosphates), has been engineered at Cys292 close to the dimer interaction area. This residue was earlier shown to be specifically photoaffinity labelled with the allosteric nucleotide dTTP. In this study the effect of the Cys292-->Ala substitution is shown to be an overall diminished nucleotide binding at the specificity site reflected in Kd values for dTTP, dGTP and dATP higher by more than one order of magnitude with respect to wild type. The mutant protein's interaction with other protein components of the ribonucleotide reductase system was unaffected by the mutation. These results show that Cys292 in protein R1 of class I ribonucleotide reductase from E. coli is located in the allosteric specificity site.