Role of MgATP in the activation and reassociation of cAMP-dependent protein kinase I: consequences of replacing the essential arginine in cAMP binding site A.
The type I form of cAMP-dependent protein kinase binds MgATP with a high affinity, and binding of MgATP decreases the affinity of the holoenzyme for cAMP [Hofmann et al. (1975) J. Biol. Chem. 250, 7795]. Holoenzyme was formed here with a mutant form of the bovine recombinant type I regulatory subunit where the essential arginine in site A, Arg-209, was replaced with Lys. Although this mutation does not significantly change the high-affinity binding of MgATP to the holoenzyme, it does abolish high-affinity binding of cAMP to site A. In the absence of MgATP, binding of cAMP to site B is sufficient to promote dissociation of the holoenzyme complex and activation of the catalytic subunit [Bubis et al. (1988) J. Biol. Chem. 263, 9668]. In the presence of MgATP however, holoenzyme formed with this mutant regulatory subunit is very resistant to cAMP. The Kd(cAMP) was greater than 1 microM, and the Ka(cAMP) increased 60-fold from 130 nM to 6.5 microM in the presence of MgATP. Thus, MgATP serves as a lock that selectively stabilizes the holoenzyme and inhibits activation. Both site A and site B are shielded from cAMP in the presence of MgATP. These results suggest that Arg-209 may play a role in stabilizing the MgATP.holoenzyme complex in addition to its role in binding the exocyclic oxygens of cAMP when cAMP is bound to the regulatory subunit. The catalytic subunit also reassociates rapidly with this mutant regulatory subunit, and in contrast to the wild-type regulatory subunit, holoenzyme formation does not require MgATP.
Neitzel
J J
JJ
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Dostmann
W R
WR
Taylor
S S
SS
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001120:Arginine / Q000235:genetics*; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D011494:Protein Kinases / Q000378:metabolism*
D000255:Adenosine Triphosphate; D001120:Arginine; D000242:Cyclic AMP; D011494:Protein Kinases