Zorn M, Fladmark K E, Ogreid D, Jastorff B, Døskeland S O, Dostmann W R
Institut für Pharmakologie und Toxikologie, Technische Universität München, Germany.
FEBS Lett. 1995 Apr 10;362(3):291-4. doi: 10.1016/0014-5793(95)00261-7.
A single amino acid substitution (Ala335Asp) in cAMP binding site B of the regulatory subunit of cAMP-dependent protein kinase type I was sufficient to abolish high affinity cAMP binding for both cAMP binding sites A and B. Furthermore, the Ala335Asp mutation increased the activation constant for cAMP of the mutant holoenzyme 30-fold and also enhanced the rate of holoenzyme formation. Thus, the substitution was responsible for the dominant negative phenotype of the enzyme. Activation of mutant holoenzyme with site-selective cAMP analogs indicated that the enzyme dissociated through binding to site A only. Our results provide evidence that Ala335 is an essential residue for high affinity cAMP binding of both sites as well as for the functional integrity of the enzyme.
