title: Ala335 is essential for high-affinity cAMP-binding of both sites A and B of cAMP-dependent protein kinase type I.
abstract: 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.
authors.affiliation: Institut für Pharmakologie und Toxikologie, Technische Universität München, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Zorn
authors.forename: K E
authors.initials: KE
authors.lastname: Fladmark
authors.forename: D
authors.initials: D
authors.lastname: Ogreid
authors.forename: B
authors.initials: B
authors.lastname: Jastorff
authors.forename: S O
authors.initials: SO
authors.lastname: Døskeland
authors.forename: W R
authors.initials: WR
authors.lastname: Dostmann
mesh_terms: D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine