Adenosine-3′,5′-Monophosphate-Dependent Protein Kinase From Bovine Anterior Pituitary Gland. III. Structural Specificity of the ATP Site of the Catalytic Subunit

The effect of analogues and derivatives of adenosine 3′,5′-monophosphate (cyclic AMP) and of ATP on the incorporation of 32P from [γ-32P]ATP into histones has been measured using the purified catalytic subunit of adenohypophyseal protein kinase. Gamma-labeled CTP, GTP, and UTP cannot substitute for [γ-32P]ATP but they slightly inhibit the phosphorylation by [γ-32P]-ATP when present as unlabeled compounds. A stringent requirement of the adenine nucleus is observed for the ability to compete at the ATP site, inhibitions of 42, 39, 32, and 63% being observed respectively with adenine, adenosine, 5′AMP, and ADP, while the corresponding purine or pyrimidine derivatives have no effect when present at a 13-fold molar excess relative to [γ-32P]ATP. The N6-benzoyl and N6-butyryl derivatives of cyclic AMP are inactive whereas the 8-substituted derivatives are generally as active as cyclic AMP itself, except for the 8-amino- and 8-hydroxy-derivatives, which exhibit a lower degree of competition. All cyclic AMP and ATP analogues and derivatives that inhibit histone phosphorylation by [γ-32P]ATP act as competitive inhibitors. Such competition at the ATP site of the catalytic subunit of protein kinase probably accounts for the progressive inhibition of cyclic-AMP-dependent protein kinase activity measured at high concentrations (above 10−5 M) of the cyclic nucleotide.