Ala335 is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

FEBS Letters

Døskeland, Stein O.²  Zorn, Michaele¹  Dostmann, Wolfgang R.G.¹  Fladmark, Kari E.⁴  Øgreid, Dagfinn⁴  Jastorff, Bernd³ 

[1] Institut für Pharmakologie und Toxikologie, Technische Universität München, Biedersteiner Str. 29, 80802 München, Germany;Institute of Anatomy and Cell Biology, University of Bergen, Årstadveien 19, 5009 Bergen, Norway;Institut für Organische Chemie, Universität Bremen, Leobener Str., NW 2, 28359 Bremen, Germany;Center of Molecular Medicine, Haukeland Hospital, University of Bergen, 5021 Bergen, Norway

关键词: Cyclic AMP-dependent protein kinase;    cAMP analog;    Site-directed mutagenesis;   

DOI  :  10.1016/0014-5793(95)00261-7

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A single amino acid substitution (Ala³³⁵ 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 Ala³³⁵ 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 Ala³³⁵ is an essential residue for high affinity cAMP binding of both sites as well as for the functional integrity of the enzyme.

【 授权许可】

Unknown