Salt-Induced Conformational Changes in the Catalytic Subunit of Adenosine Cyclic 3′,5′-Phosphate Dependent Protein Kinase. Use for Establishing a Connection between One Sulfhydryl Group and the γ-P Subsite in the ATP Site of This Subunit

The sulfhydryl groups in the catalytic subunit (C) of cAMP-dependent protein kinase are shown to behave as built-in reporter groups, whose relative chemical reactivity can be used to demonstrate that C readily undergoes a salt-induced conformational change at neutral pH and around physiological values of ionic strength. Upon increasing the ionic strength of the medium from 0.03 to 0.22, one SH group in C becomes more reactive toward 5,5′-dithiobis(2-nitrobenzoic acid) (the rate constant increases ∼4.5-fold) while the other SH group in C becomes less reactive toward the same reagent (the rate constant decreases ∼3.8-fold). Modification of the SH groups of C by this reagent brings about an inactivation of the enzyme which, at low ionic strength, can be shown to occur concomitantly and stoichiometrically with the modification of one (kinetically characterized) sulfhydryl. When ATP and its analogues are used to protect the enzyme from inactivation by this reagent, a connection is established between this SH group and the γ-P subsite of the ATP binding site in C. In parallel with the above-mentioned salt-induced conformational change, the C subunit undergoes an inactivation (which increases with ionic strength) as measured by histone H2b phosphorylation. Though not reflected in the V_max, this conformational change considerably increases the K_m of the enzyme for histone H2b (∼4-fold) as well as for MgATP (∼3.4-fold). This intrinsic malleability of the enzyme, shown here to occur even in the absence of substrate, can account for the well-known salt inhibition of the enzyme for certain substrates and the ion-dependent activation toward other substrates. It can also account for the somewhat contradictory results reported from different laboratories with regard to the functional role of the sulfhydryl groups in the enzyme. It is suggested that this intrinsic malleability might constitute a molecular basis for modulating the specificity of the enzyme and targeting its activity from one substrate to another in response to intracellular specifier signals.