A CGMP KINASE MUTANT WITH INCREASED SENSITIVITY TO THE PROTEIN-KINASEINHIBITOR PEPTIDE PKI(5-24)

Synthetic peptides corresponding to the active domain of the heat-stab le inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent pr otein kinase. In this study, we tried to confer PKI sensitivity to cGM P kinase by site-directed mutagenesis, The molecular requirements for high affintiy inhibition by PKI were deduced from the crystal structur e of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr(235) and Phe(239) in the catalytic subunit, which from a sandwich-like structure with Phe(10) of the PKI(5-24) peptide, To incr ease the sensitivity for PKI, the cGMP kinase codons at the correspond ing sites, Ser(555) and Ser(559), were changed to Tyr and Phe, The mut ant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM), Wild type and mut ant cGMP kinase did not differ significantly in their K-m and v(max) f or three different substrate peptides, The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher pot ency than that of wild type, with K-i values of 42 +/- .3 mu M and 160 +/- .7 mu M, respectively, The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide, Mutation of the essenti al Phe(10) in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with K-i values of 160 +/- 11 and 169 +/- 27 mu M, respectively, These resu lts suggest that the mutations Ser555Tyr and Ser559Phe are required, b ut not sufficient, for high affinity inhibition of cGMP kinase by PKI.