Both cyclic GMP-dependent protein kinase (cGK) and cyclic AMP-dependent protein kinase (cAK) contain two distinct cyclic nucleotide-binding sites referred to as fast and slow sites based on cyclic nucleotide dissociation behavior. In cAK, the fast site lies amino-terminal to the slow site, and sequence homologies between cAK and cGK have suggested similar positioning for the sites in cGK. Recombinant human type I␤ cGK (wild type (WT) cGK) was overexpressed, and the properties of purified WT cGK and native type I␤ cGK were similar. cGK was mutated singly at Thr-193 (T193A, T193V, and T193S) and Thr-317 (T317A, T317V, and T317S), which have been predicted to provide cGMP specificity in the cGMP-binding sites of cGK; a double mutant (T193A/ T317A) was produced also. Compared with WT cGK, half-maximal activation (K a ) of mutant cGKs by cGMP was increased 2-(T317A), 27-(T193A), or 63-fold (T193A/ T317A), but the K a for cAMP of these mutants was essentially unchanged. The T193A and T193V mutants had a large increase in the rate of the slow component of The cGMP-dependent protein kinase (cGK) 1 and cAMP-dependent protein kinase (cAK) are homologous enzymes that are preferentially activated by cGMP and cAMP (1), respectively, although cross-activation of either cGK by cAMP (2) or cAK by cGMP (3) has been demonstrated under physiological and pathological conditions. The two cyclic nucleotide-binding sites in cAK preferentially bind cAMP over cGMP with a Ͼ50-fold selectivity, have different cAMP analog specificities, and have different dissociation rates for cAMP (4 -7). Because of the latter feature, these cAMP-binding sites generally have been referred to as the fast and slow cAMP-dissociation sites (see Footnote 2 of Ref. 8). Biochemical studies of type II cAK first suggested that the more amino-terminal cyclic nucleotide-binding site is the fast site whereas the adjacent site is the slow site (9), and similar approaches were used to demonstrate the same structural order for the binding sites of type I cAK (10). Molecular modeling (11) and results of site-directed mutagenesis of the binding sites (12, 13) supported these findings.Like cAK, each subunit of cGK (Fig. 1, top) contains two cyclic nucleotide-binding sites that are homologous in sequence but have distinct kinetic characteristics. The cyclic nucleotidebinding sites of cGK preferentially bind cGMP over cAMP with a Ͼ100-fold selectivity, have different cGMP analog specificities, and have extremely different dissociation rates for cGMP (14). Additionally, these sites show significant amino acid sequence homology to the two kinetically distinct cAMP-binding sites of cAK. The more amino-terminal site of cGK shows higher homology to the more amino-terminal site of cAK, and conversely, the more carboxyl-terminal site of cGK shows higher homology to the more carboxyl-terminal site of cAK (15). Based solely on the sequence homology between the cyclic nucleotide-binding sites in cAK and cGK, it has been thought that the more amino-terminal cGMP-binding site of cGK is...

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The role of each of the two different cGMP-binding sites (referred to as slow and fast sites) of type I cGMPdependent protein kinase (PKG) in altering the rate of catalysis of phosphorylation of exogenous substrates (heterophosphorylation) or the rate of autophosphorylation has not been resolved. In the present study, the cGMP concentration required for half-maximal activation (A 50 ) of wild-type PKG type I␤ (WT) was 5-fold higher for heterophosphorylation than for autophosphorylation. cGMP occupation of the slow site was associated with an increase in the autophosphorylation rate, whereas occupation of the fast and slow site together was associated with a decrease in the autophosphorylation rate compared with the rate observed with occupation of the slow site alone. The contributions of each cGMP-binding site were investigated using PKG mutants containing substitutions of an invariant threonine residue that is critical for high affinity cGMP-binding in each site. Site-directed mutagenesis of Thr-317 of the fast site (T317A) increased the cGMP A 50 for heterophosphorylation 4-fold at 30°C, with nominal effect on cGMP A 50 for autophosphorylation compared with WT. The analogous slow site mutation (T193A) increased the cGMP A 50 for heterophosphorylation and autophosphorylation 32-and 64-fold, respectively. Compared with WT, the cGMP A 50 of the double mutant (T193A/T317A) for heterophosphorylation was increased 300-fold, whereas the cGMP A 50 for autophosphorylation was similar to that of T193A. Thus, occupation of both cGMPbinding sites of PKG is required for maximal stimulation of heterophosphorylation, whereas occupation of the slow site alone is sufficient for stimulation of the rate of autophosphorylation, and additional occupation of the fast site reduces this rate.Several regulatory roles have been ascribed to cGMP-dependent protein kinase (PKG) 1 in mammalian cells (1-5).Three mammalian isozymic forms of PKG have been reported: type I␣, type I␤, and type II; each of these exists as a homodimer of subunits that contain a regulatory domain and a catalytic domain in a single polypeptide sequence. The regulatory portions of the type I enzymes comprise the amino-terminal ϳ50% of the polypeptide and contain an autoinhibitory domain, autophosphorylation sites, and two cyclic nucleotidebinding sites (Fig. 1A). The primary sequences of the types I␣ and I␤ PKG isoforms differ only in their amino-terminal segment of ϳ100 residues, sharing 36% identity in this region (6), which contains the dimerization domain, autophosphorylation sites, and autoinhibitory domain. The cGMP-binding sites in types I␣ and I␤ PKG have identical amino acid sequences but exhibit different kinetic properties and cyclic nucleotide analog specificities (7). These intramolecular binding sites are distinguished from each other by their relative affinities for cGMP, and they are described as the low affinity cGMP-binding site (fast dissociating site or fast site) and the high affinity cGMPbinding site (slow dissociating site or slow site) (8)...

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