Snapping of the carboxyl terminal tail of the catalytic subunit of PKA onto its core: characterization of the sites by mutagenesis.
A set of 45 mutants of the carboxyl terminal tail of the PKA catalytic subunit was prepared and used to assess the contribution of this tail to the structure and function of the kinase. Ala substitutions of Asp 323, Phe 327, Glu 333, and Phe 350 resulted in a complete loss of enzymatic activity. Other replacements by Ala (Phe 314, Tyr 330, Glu 332, and Phe 347) brought about either a drop in activity to less than 10% of the wild-type enzyme or a reduction of affinity toward ATP (Lys 317, Lys 319, Tyr 330, and Glu 332) or toward Kemptide (Ile 315, Tyr 330, Val 337, Ile 339, Lys 345, and Glu 346). Mutations of Ser 338, a major autophosphorylation site of PKA, by Ala, Glu, Asp, Gln, and Asn showed that the kinetic parameters of these mutants are similar to those of the wild-type. The contribution of each of these tail mutations to the structure and stability of the kinase was assessed by monitoring its effect on the heat stability (when measurable) or by determining the susceptibility of the mutant kinase to cleavage by the Kinase Splitting Membranal Proteinase/Meprin beta. Here we show that the tail of PKA has a key role in creating the active conformation of the kinase. It does so by means of specific amino acid residues, which act as "snapping points" to embrace the two lobes of the kinase and orient them in the correct juxtaposition for substrate docking, biorecognition, and catalysis.
Batkin
M
M
Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel.
Schvartz
I
I
Shaltiel
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D009842:Oligopeptides; D011994:Recombinant Proteins; C037642:kemptide; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D008666:Metalloendopeptidases; C095663:meprin B