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  • Title: 8-(2-Carboxymethylthio)-cGMP, a site-1-selective compound for cGMP-dependent protein kinase.
    Author: Landgraf W, Rack M, Heil WG, Hofmann F.
    Journal: Eur J Biochem; 1988 Mar 01; 172(2):439-44. PubMed ID: 2832164.
    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 degrees 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 microM, 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 microM CMT-cGMP at 4 degrees C. The apparent site 1 selectivity was 250-fold at 30 degrees C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 microM 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 microM 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 microM and 2 microM, 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 expression of a partial enzyme activity.
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