8‐(2‐Carboxymethylthio)‐cGMP, a site‐1‐selective compound for cGMP‐dependent protein kinase

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Abstract
The cGMP analogue 8-(2-carboxymethylthio)-cGMP (CMT-cGMP) was synthesized and its binding to cGMP-dependent protein kinase (cGMP kinase) was studied. CMT-cGMP bound at 4.degree. C with an over 1400-fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 .mu.M, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 .mu.M CMT-cGMP at 4.degree. C. The apparent site 1 selectivity was 250-fold at 30.degree. C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 .mu.M for site 1 and 2, respectively. CMT-cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Ka values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 .mu.M CMT-cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT-cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT-cGMP and cGMP inhibited cAMP-stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 .mu.M and 2 .mu.M, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself. These results indicate that CMT-cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expession of a partial enzyme activity.