On the Protein Accommodating Site of the Catalytic Subunit of Adenosine Cyclic 3′,5′-Phosphate Dependent Protein Kinase

The catalytic subunit (C) of cAMP-dependent protein kinase (EC 2.7.1.37) contains two kinetically characterized sulfhydryl groups SH_I and SH_II, one of which (SH_II) was previously shown to be intimately associated with the γ-P subsite of the ATP binding site in C [Jiménez, J. S., Kupfer, A., Gani, V., & Shaltiel, S. (1982) Biochemistry (preceding paper in this issue)]. In the presence of either histone H2b or protamine (two protein substrates of the enzyme) both SH_I and SH_II display a considerably enhanced reactivity toward the negatively charged thiol reagent 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs₂), with a parallel acceleration in the rate of inactivation of the kinase. By use of a series of neutral or negatively charged analogues of Nbs₂, it is shown that the enhanced chemical reactivity of the sulfhydryls occurs only with the negatively charged analogues and therefore it presumably originates from an increase in the local concentration of the reagent around the sulfhydryls, i.e., from a “channeling effect” caused by the positively charged protein substrate. In contrast, the heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly enhances the chemical reactivity of SH_II only, without affecting SH_I, but still accelerates the rate of inactivation of the enzyme. Being a good substrate of small size, the channeling effect of this peptide is limited to the active site and its vicinity, suggesting that SH_II is adjacent to the protein binding moiety of the active site. This suggestion is further supported by the finding that the regulatory subunit (R) which is known to block access to the active site in the holoenzyme affords a considerable protection from modification to the SH_II group of C and preserves the potential catalytic activity of R₂C₂. Furthermore, full shielding of SH_II and complete prevention of the loss of potential catalytic activity are achieved by addition of MgATP which (upon binding to its high-affinity site) locks the enzyme in its undissociated form. The implications of these results in terms of the position of SH_II within the active site of the enzyme and relative to the position of SH_I are discussed.