Belduz A O, Lee E J, Harman J G
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061.
Nucleic Acids Res. 1993 Apr 25;21(8):1827-35. doi: 10.1093/nar/21.8.1827.
The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions 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 substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-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-responsive 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.
将大肠杆菌环磷酸腺苷（cAMP）受体蛋白（CRP）的3'，5'环磷酸腺苷结合口袋中的谷氨酸72突变为亮氨酸、谷氨酰胺或天冬氨酸；将精氨酸82突变为赖氨酸、组氨酸、亮氨酸、异亮氨酸或谷氨酰胺。在野生型CRP以及一种CRP*（即不依赖cAMP的）蛋白形式中进行替换，以评估氨基酸替换对CRP结构的影响。通过β-半乳糖苷酶活性和cAMP结合活性的测量对含有结合口袋残基替换形式CRP的细胞进行表征。本研究证实了谷氨酸72和精氨酸82在cAMP结合及CRP激活过程中的作用。将谷氨酸72替换为谷氨酰胺或亮氨酸会产生对cAMP亲和力低且对该核苷酸无反应的CRP形式。用天冬氨酸替换谷氨酸72会产生一种低亲和力的cAMP反应性CRP形式。CRP对环核苷酸结合口袋内72位谷氨酸羧基的定位有严格要求。本研究结果还表明，cAMP与cGMP（一种cAMP与CRP结合的竞争性抑制剂）的结合要求存在差异。
