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Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (Ka) of each mutant protein for phosphotransferase activation and allosteric [3H]cGMP-binding affinity (KD) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (Km) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.