Mutations in the autoinhibitor site of the regulatory subunit of cAMP-dependent protein kinase I : replacement of Ala-97 and Ser-99 interferes with reassociation with the catalytic subunit

摘要: Each regulatory (R) subunit of cAMP-dependent protein kinase contains an autoinhibitor site that lies approximately 90-100 residues from the amino terminus. In order to study importance this in type I R-subunit for interacting with catalytic (C) subunit, recombinant techniques were used replace Ala-97 Gln, His, Lys, and Arg Ser-99 Gly Lys. All mutant proteins having a replacement at showed reduced affinity C-subunit ranging 14- 55-fold. general, decrease mutants correlated increase size side chain. contrast wild R-subunit, where MgATP facilitates holoenzyme formation, inhibits reassociation all suggesting larger chains sterically interfere bound active C-subunit. Whereas slowed AMP actually accelerated A97K, A97H (pH 6.0), A97Q Therefore, Lys-97, His-97, Gln-97 can interact either electrostatically or by hydrogen bonding phosphate AMP. This interpretation is reinforced fact stimulatory effect on was pH-dependent. The affinities S99G S99K 7- 24-fold, respectively, also may contribute interactions between R- C-subunits.