The catalytic domain of the cGMP-dependent protein kinase Ialpha modulates the cGMP-binding characteristics of its regulatory domain.
The cGMP-dependent protein kinase Ialpha (PKG Ialpha) possesses two functional moieties, the regulatory and catalytic domains, which reside on a single polypeptide chain. Here we report on the influence of the catalytic domain on the binding of cGMP to the regulatory domain. A deletion mutant, delta352-670 of PKG Ialpha, lacking the catalytic domain, was constructed and expressed in E. coli. The purified 38 kDa mutant protein showed strong reactivity toward tryptic proteolysis at residue Arg77. Thus, a double deletion fragment delta1-77/352-670 PKG Ialpha, lacking the N-terminus, was also purified. Both proteins had functional cGMP binding, but differed kinetically from the wild-type protein. First the affinity constants for cGMP were modulated, second the constructs showed no signs of cooperative cGMP binding and third dimerization of the delta352-670 mutant was abolished. Our results provide evidence that the catalytic domain forms an intimate interaction with the regulatory domain and modulates the kinetics of cGMP binding.
Dostmann
W R
WR
Institut für Pharmakologie und Toxikologie, Technische Universität München, Germany. dostmann@ipt.med.tu-muenchen.de
Koep
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Endres
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D001665:Binding Sites; D006152:Cyclic GMP / Q000378:metabolism*; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP