Ala335 is essential for high-affinity cAMP-binding of both sites A and B of cAMP-dependent protein kinase type I.
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.
Zorn
M
M
Institut für Pharmakologie und Toxikologie, Technische Universität München, Germany.
Fladmark
K E
KE
Ogreid
D
D
Jastorff
B
B
Døskeland
S O
SO
Dostmann
W R
WR
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
D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000409:Alanine