Abstract
The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels.
Highlights
TheCAMP-dependent protein kinase contains two signaling pathways mainly through changes in their cyclic different CAMP-binding sites referred to as the slow nucleotide-binding characteristics
The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase
Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP
Summary
Mutation of Ala-334 to a threonine in latory component contains two cyclic nucleotide-binding dothe slow site of the bovine type I regulatory subunit mains that are more divergent between each other than are created a site with marked increase in cGMP affinity the corresponding domains between kinase isozymes [4, 8]. Mutation of Ala-334 to a threonine in the slow site’of type ICAMP-dependentprotein kinase regulatory (R) subunit created abinding site with a marked increase in cGMP affinity without altering cAMP affinity [14]. Protein Kinuse Activation-Activation constants (K.) were meas- 0greid et al [22] have noted a direct correlation between the K', ured for holoenzymes formed by combining wild-type and mutant R and K I A B , based on extensive analog studies of native type I cAMP subunits with bovine heart C subunitas described previously [14]. Was quantitated by the phosphocellulose paper method of Roskoski [20]. cAMP Dissociation Rates
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