The negative charge of Glu‐127 in protein kinase A and its biorecognition

FEBS Letters

Batkin, Michael¹  Shaltiel, Shmuel¹ 

[1] Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel

关键词: Protein kinase A;    Site-directed mutagenesis;    Active site;    Biorecognition;    Specificity;    Ionic interactions;   

DOI  :  10.1016/S0014-5793(99)00500-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A set of mutants of protein kinase A (PKA) in which Glu-127 was replaced by Gln, Asp, Asn, and Arg was prepared. Their K _m and V _max values show that the negative charge of Glu-127 (not merely its hydrogen bonding capacity) is indispensable for the kinase activity, since Glu-127/Gln is inactive, in spite of the fact that it can form hydrogen bonds and is very similar in bulkiness and conformation to wt-PKA. Glu-127 is involved in the biorecognition of PKA, interacting ionically with the positively charged guanido group of Arg P-3 (a major recognition element in the consensus sequence of PKA). In support of this conclusion, it is shown that a regression of the Glu-127 carboxylate by 1.54 Å (as in Glu-127/Asp) results in an active kinase with a similar thermal stability and susceptibility to conformation-dependent proteolysis, a similar V _max, an identical K _m for ATP, but a >20-fold higher K _m for kemptide. The two inactive mutants of PKA, Glu-127/Gln and Glu-127/Asn, are potentially useful for studying protein-protein interactions of PKA, e.g. for monitoring enzymatically the displacement of active PKA from its complexes.

【 授权许可】

Unknown