ALA(335) IS ESSENTIAL FOR HIGH-AFFINITY CAMP-BINDING OF BOTH SITE-A AND SITE-B OF CAMP-DEPENDENT PROTEIN-KINASE TYPE-I

作者：ZORN M; FLADMARK KE; OGREID D; JASTORFF B; DOSKELAND SO; DOSTMANN WRG

来源：FEBS Letters, 1995, 362(3): 291-294.

DOI：10.1016/0014-5793(95)00261-7

• CYCLIC AMP-DEPENDENT PROTEIN KINASE
• CAMP ANALOG
• SITE-DIRECTED MUTAGENESIS

A single amino acid substitution (Ala(335)Asp) 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 Ala(335)Asp 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 Ala(335) is an essential residue for high affinity cAMP binding of both sites as well as for the functional integrity of the enzyme.

• 出版日期1995-4-10