Holoenzyme interaction sites in the cAMP-dependent protein kinase. Histidine 87 in the catalytic subunit complements serine 99 in the type I regulatory subunit.

摘要: Two mutations of the catalytic (C) subunit cAMP-dependent protein kinase where His87 was changed to Ala and Asp were expressed in Escherichia coli purified. These mutants phosphorylated at Thr197 catalytically active, although some changes their kinetic parameters observed. The most striking differences interaction with physiological inhibitors. Both inhibited by inhibitor Ki values below 50 nM. defective type I regulatory (RI) as measured (i) rate holoenzyme formation cAMP bound RI-subunit (ii) apparent Kd cAMP-free RI-subunit. impaired both presence absence ATP His mutant showing greatest effect. C-subunits also combined RI-subunits that contained autoinhibitor sequence Arg94 (P-3) Ser99 (P + 2). Complementarity between established, but not Arg94. Holoenzyme a Ser99-->Lys less dependent on when either C-subunit than it wild-type C-subunit. for combinations measured. compensated mutants. His87-->Ala unable form an complex which binding A-site. This indicated this R-subunit recognition well binding. roles Arg209 R-C interactions are discussed.