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A single amino acid substitution (Ala33SAsp) 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 Ala33SAsp 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.B has been shown to interact with the adenine moiety since alteration of this residue changed the affinity and specificity for cGMP [13]. Recently, a sub-clone of the rat myelocytic leukemia cell line (IPC-81) has been found to harbor a heterozygously expressed point mutation [14]. This mutated clone was selected by treatment with 8-CPT-cAMP and identified as Alato-Asp at the invariant position 335 [14]. In order to study the effects of this mutation on ligand binding and holoenzyme activation we used recombinant protein expression techniques to obtain large quantities of pure recombinant Ala335Asp mutant protein. Materials and methods

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