MUTAGENESIS OF THE CYCLIC-AMP RECEPTOR PROTEIN OF ESCHERICHIA-COLI - TARGETING POSITIONS 72 AND 82 OF THE CYCLIC-NUCLEOTIDE BINDING POCKET

The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to su bstitute leucine, glutamine, or aspartate for glutamate 72; and lysine , histidine, leucine, isoleucine, or glutamine for arginine 82. Substi tutions were made in wild-type CRP and in a CRP, or cAMP-independent, form of the protein to assess the effects of the amino acid substitut ions on CRP structure. Cells containing the binding pocket residue-sub stituted forms of CRP were characterized through beta-galactosidase ac tivity and by measurement of cAMP binding activity. This study confirm s a role for both glutamate 72 and arginine 82 in cAMP binding and act ivation of CRP. Glutamine or leucine substitution of glutamate 72 prod uced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group withi n the cyclic nucleotide binding pocket. Results of this study also ind icate that there are differences in the binding requirements of cAMP a nd cGMP, a competitive inhibitor of cAMP binding to CRP.