Nucleic Acids Research, 1993, Vol. 21, No. 8 1827-1835
© 1993
MOLECULAR BIOLOGY |
Mutagenesis of the cyclic AMP receptor protein of Escherichia coli targeting positions 72 and 82 of the cyclic nucleotide binding pocket
Department of Chemistry and Biochemistry, Texas Tech University Lubbock, Tx 79409-1061, USA
* To whom correspondence should be addressed
Received January 11, 1993. Revised March 12, 1993. Accepted March 12, 1993.
The 3', 5' cyclic adenoslne monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escheiichla coll was mutagenlzed to substitute leuclne, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, Isoleuclne, or glutamine for arginine 82. Substitutions were made In wild-type CRP and In a CRP*, or cAMP-lndependent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through ß-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responslve form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also Indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive Inhibitor of cAMP binding to CRP.
* Present address: Karadeniz Technical University, Department of Biology, 61080-Trabzon, Turkey
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