Is there a catalytic base in the active site of cAMP-dependent protein kinase?
The carboxyl group of an aspartic acid in the active site of the serine-specific protein kinase, cAMP-dependent protein kinase, is poised near the hydroxyl proton of a peptide substrate in the X-ray crystallographic structure (Madhusudan et al., 1994), suggesting that this residue may act as a general-base catalyst in the phosphoryl transfer reaction. Indeed, several proposals have been made in this regard. We measured the pre-steady-state kinetics in this enzyme using a rapid quench flow technique to understand the role of this putative base. The phosphorylation of the peptide substrate, GRTGRRNSI, by cAMP-dependent protein kinase exhibited "burst" kinetics consistent with a mechanism in which the peptide is phosphorylated rapidly (154 s(-1)) and the product(s) is (are) released slowly (16 s(-1)). The replacement of Mg2+ with Mn2+ leads to a 13-fold reduction in this observed "burst" rate constant, suggesting that this transient is limited either by the phosphoryl transfer step or by a metal ion-dependent conformational change step. The influence of deuterium oxide on the pre-steady-state kinetics was monitored in the presence of both divalent metal ions, and no solvent isotope effect was measured on either "burst" phase. A large solvent isotope effect is observed on k(cat) in the presence of either metal ion, and a proton inventory analysis in the presence of Mg2+ indicates that two or more protons are transferred in the product release step. Finally, no pH dependence is observed on the "burst" rate constant using either Mg2+ or Mn2+ over the pH range of 6-9. The combined data do not support a mechanism involving a general-base catalyst whose pK(a) is greater than 5 or less than 10 if the "burst" phase is cleanly limited by the phosphoryl transfer step. If the "burst" phase is limited by a metal ion-dependent conformational change step, the measurement of the phosphoryl transfer step is obscured, and the participation of a base catalyst is indeterminate.
Zhou
J
J
Department of Chemistry, San Diego State University, California 92182-1030, USA.
Adams
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008956:Models, Chemical; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012997:Solvents; D014783:Viscosity
D010455:Peptides; D011994:Recombinant Proteins; D012997:Solvents; D008345:Manganese; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium