The catalytic subunit (C) of cAMP-dependent protein kinase (EC 220.127.116.11) contains two kinetically characterized sulfhydryl groups SHI and SHII, one of which (SHII) 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 SHI and SHII 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 SHII only, without affecting SHI, 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 SHII 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 SHII group of C and preserves the potential catalytic activity of R2C2. Furthermore, full shielding of SHII 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 SHII within the active site of the enzyme and relative to the position of SHI are discussed.
ASJC Scopus subject areas