Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2

Biochim Biophys Acta. 2002 Jul 29;1598(1-2):88-97. doi: 10.1016/s0167-4838(02)00340-0.


The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Cloning, Molecular
  • Enzyme Activation
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Kinetics
  • Peptide Fragments / chemistry
  • Peptide Mapping
  • Phosphorylation
  • Protein Serine-Threonine Kinases / chemistry
  • Protein Serine-Threonine Kinases / isolation & purification
  • Protein Serine-Threonine Kinases / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Substrate Specificity


  • Intracellular Signaling Peptides and Proteins
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • MAP-kinase-activated kinase 2
  • Protein Serine-Threonine Kinases