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Divalent metal ions influence catalysis and active‐site accessibility in the camp‐dependent protein kinase
Author(s) -
Adams Joseph A.,
Taylor Susan S.
Publication year - 1993
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560021217
Subject(s) - chemistry , divalent , reaction rate constant , substrate (aquarium) , product inhibition , metal , dissociation constant , protein kinase a , metal ions in aqueous solution , active site , peptide , catalysis , stereochemistry , kinetics , enzyme , non competitive inhibition , biochemistry , biology , organic chemistry , receptor , physics , quantum mechanics , ecology
Abstract Phosphorylation of the peptide LRRASLG by the catalytic subunit of cAMP‐dependent protein kinase was measured in the presence of various divalent metals to establish the role of electrophiles in the kinetic mechanism. Under conditions of low or high metal concentrations, the apparent second‐order rate constant, k cat / K peptide , and the maximal rate constant, k cat , followed the trend Mg 2+ > Co 2+ > Mn 2+ . Competitive inhibition studies indicate that the former effect is not due to destabilization of the substrate complex, E‐ATP‐S. The effects of solvent viscosity on the steady‐state kinetic parameters were interpreted according to a simple mechanism involving substrate binding, phosphotransfer, and product release steps and two metal chelation sites in the nucleotide pocket. Decreases in k cat and k cat / K peptide result mostly from attenuations in the dissociation rate constant for ADP and the association rate constant for the substrate, respectively. Decreases in the phosphoryl transfer rate constant have only negligible to moderate effects on these parameters. The low observed values for the association rate constant of the substrate indicate that the metals control the concentration of the productive binary form, E a ‐ATP, and indirectly the accessibility of the active site. By comparison, Mg 2+ is the best divalent metal catalyst because it uniformly lowers the transition state energies for all steps in the kinetic mechanism, permitting maximum flux of substrate to product. The data suggest that cAMP‐dependent protein kinase uses metal ions to serve multiple roles in facilitating phosphotransfer and accelerating substrate association and product dissociation.