Identification of the amino acid sequences responsible for high affinity activation of cGMP kinase Ialpha.
The cGMP-dependent protein kinases (cGK) Ialpha and Ibeta have identical cGMP binding sites and catalytic domains. However, differences in their first 100 amino acids result in 15-fold different activation constants for cGMP. We constructed chimeras to identify those amino acid sequences that contribute to the high affinity cGK Ialpha and low affinity cGK Ibeta phenotype. The cGK Ialpha/Ibeta chimeras contained permutations of six amino-terminal regions (S1-S6) including the leucine zipper (S2), the autoinhibitory domain (S4), and the hinge domain (S5, S6). The exchange of S2 along with S4 switched the phenotype from cGK Ialpha to cGK Ibeta and vice versa, suggesting that the domains with the highest homology between the two isozymes determine their affinity for cGMP. The high affinity cGK Ialpha phenotype was also obtained by a specific substitution within the hinge domain. Chimeras with the sequence of cGK Ialpha in S5 and cGK Ibeta in S6 were activated at up to 6-fold lower cGMP concentrations than cGK Ialpha. Based on the activation constants of all chimeras constructed, empirical weighting factors have been calculated that quantitatively describe the contribution of the individual amino-terminal domains S1-S6 to the high affinity cGK Ialpha phenotype.
Ruth
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Institut für Pharmakologie und Toxikologie der Technische Universität München, Biedersteiner Strasse 29, D-80802 München, Germany. ruth@ipt.med.tu-muenchen.de
Pfeifer
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A
Kamm
S
S
Klatt
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Dostmann
W R
WR
Hofmann
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F
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D016350:Leucine Zippers; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013737:Testis / Q000201:enzymology
D011993:Recombinant Fusion Proteins; D010770:Phosphotransferases; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases