Dissection of the nucleotide and metal-phosphate binding sites in cAMP-dependent protein kinase

The catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is more s table by several criteria when it is part of a holoenzyme complex. By measu ring the thermal stability of the free C subunit in the presence and absenc e of nucleotides and/or divalent metal ions, it was found that most of the stabilizing effects associated with the type I holoenzyme could be attribut ed to the nucleotide. The specific requirements for this enhanced stability were further dissected: Adenosine stabilized the C subunit up to 5 degrees C; however, divalent cations (i.e., Mg2+, Ca2+, and Mn2+) do not increase heat stability in combination with adenosine and adenine (1). Divalent cati ons as well as ATP and ADP have no effect by themselves (2). The enhanced s tability derived from both ATP and ADP requires divalent cations. MnATP (12 degrees C) shows a much stronger effect than CaATP (7 degrees C) and MgATP (5 degrees C) (3). In the holoenzyme complex or the protein kinase inhibit or/C subunit complex, metal/ATP is also required for enhanced stability; ne ither the RI or RII subunits nor PKI alone stabilize the C subunit signific antly (4). For high thermal stability, the occupation of the second, low-af finity metal-binding site is necessary (5). From these results, we conclude d that the adenine moiety works independently from the metal-binding sites, stabilizing the free C subunit by itself. When the beta- and gamma-phospha tes are present, divalent metals are required for positioning these phospha tes, and two metals are required to achieve thermostability comparable to a denosine alone. The complex containing two metals is the most stable. A com parison of several conformations of the C subunit derived from different cr ystal structures is given attributing open and closed forms of the C subuni t to less and more thermostable enzymes, respectively.