Open AccessA catalytically active fragment of cGMP‐dependent protein kinase
Author(s) -
HEIL Wolfgang G.,
LANDGRAF Wolfgang,
HOFMANN Franz
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb13395.x
Subject(s) - autophosphorylation , phosphotransferase , enzyme , biochemistry , trypsin , chemistry , binding site , protein kinase a , active site , biology , stereochemistry
Treatment of cGMP‐dependent protein kinase with low concentrations of trypsin generates an enzyme fragment of 65 kDa which is fully active in the absence of cGMP. The fragment has a s 20,w value of 4.6 S indicating that the active fragment is a monomer of 65 kDa. Trypsin removes the first 77 amino acids which contain the aminoterminal dimerization site and the autophosphorylation sites. The K m and V max values of the fragment for ATP and Kemptide were essentially the same as those for the native enzyme. The fragment binds 2 mol cGMP/mol fragment with affinities close to that of the native enzyme. However, binding of cGMP to these sites was non‐cooperative and shows similar characteristics to the autophosphorylated native enzyme. These results indicate that the aminoterminal dimerization site of cGMP‐dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.