IDENTIFICATION OF THE AMINO-ACID-SEQUENCES RESPONSIBLE FOR HIGH-AFFINITY ACTIVATION OF CGMP KINASE I-ALPHA

The cGMP-dependent protein kinases (cGK) I alpha and I beta have ident ical cGMP binding sites and catalytic domains, However, differences in their first 100 amino acids result in 15-fold different activation co nstants for cGMP, We constructed chimeras to identify those amino acid sequences that contribute to the high affinity cGK I alpha and low af finity cGK I beta phenotype, The cGK I alpha/I beta chimeras contained permutations of six amino-terminal regions (S1-S6) including the leuc ine zipper (S2), the autoinhibitory domain (S4), and the hinge domain (S5, S6). The exchange of S2 along with S4 switched the phenotype from cGK I alpha to cGK I beta and vice versa, suggesting that the domains with the highest homology between the two isozymes determine their af finity for cGMP, The high affinity cGK I alpha phenotype was also obta ined by a specific substitution within the hinge domain, Chimeras with the sequence of cGK I alpha in S5 and cGK I beta in S6 were activated at up to 6-fold lower cGMP concentrations than cGK I alpha. Based on the activation constants of all chimeras constructed, empirical weight ing factors have been calculated that quantitatively describe the cont ribution of the individual amino-terminal domains S1-S6 to the high af finity cGK I alpha phenotype.