THE CATALYTIC DOMAIN OF THE CGMP-DEPENDENT PROTEIN-KINASE I-ALPHA MODULATES THE CGMP-BINDING CHARACTERISTICS OF ITS REGULATORY DOMAIN

The cGMP-deficient protein kinase I alpha (PKG I alpha) possesses two functional moieties, the regulatory and catalytic domains, which resid e on a single polypeptide chain, Here ne report on the influence of th e catalytic domain on the binding of cGMP to the regulatory domain. A deletion mutant, Delta 352-670 of PKG I alpha lacking tile catalytic d omain, was constructed and expressed in E. coli. The purified 38 kDa m utant protein showed strong reactivity toward tryptic proteolysis at r esidue Arg(77). Thus, a double deletion fragment Delta 1-77/352-670 PK G I alpha, lacking the N-terminus, was also purified, Both proteins ha d 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 Delta 352-670 mutant was abolished, Our results pr ovide evidence that the catalytic domain forms an intimate interaction with the regulatory domain and modulates the kinetics of cGMP binding .