Kinetic mechanism for p38 MAP kinase α

p38 Mitogen‐activated protein kinase alpha (p38 MAPKα) is a member of the MAPK family. It is activated by cellular stresses and has a number of cellular substrates whose coordinated regulation mediates inflammatory responses. In addition, it is a useful anti‐inflammatory drug target that has a high specificity for Ser‐Pro or Thr‐Pro motifs in proteins and contains a number of transcription factors as well as protein kinases in its catalog of known substrates. Fundamental to signal transduction research is the understanding of the kinetic mechanisms of protein kinases and other protein modifying enzymes. To achieve this end, because peptides often make only a subset of the full range of interactions made by proteins, protein substrates must be utilized to fully elucidate kinetic mechanisms. We show using an untagged highly active form of p38 MAPKα, expressed and purified from Escherichia coli [Szafranska AE, Luo X & Dalby KN (2005) Anal Biochem 336 , 1–10) that at pH 7.5, 10 m m Mg 2+ and 27 °C p38 MAPKα phosphorylates ATF2Δ115 through a partial rapid‐equilibrium random‐order ternary‐complex mechanism. This mechanism is supported by a combination of steady‐state substrate and inhibition kinetics, as well as microcalorimetry and published structural studies. The steady‐state kinetic experiments suggest that magnesium adenosine triphosphate (MgATP), adenylyl (β,γ‐methylene) diphosphonic acid (MgAMP‐PCP) and magnesium adenosine diphosphate (MgADP) bind p38 MAPKα with dissociation constants of K A  = 360 µ m , K I  = 240 µ m , and K I  > 2000 µ m , respectively. Calorimetry experiments suggest that MgAMP‐PCP and MgADP bind the p38 MAPKα–ATF2Δ115 binary complex slightly more tightly than they do the free enzyme, with a dissociation constant of K d  ≈ 70 µ m . Interestingly, MgAMP‐PCP exhibits a mixed inhibition pattern with respect to ATF2Δ115, whereas MgADP exhibits an uncompetitive‐like pattern. This discrepancy occurs because MgADP, unlike MgAMP‐PCP, binds the free enzyme weakly. Intriguingly, no inhibition by 2 m m adenine or 2 m m MgAMP was detected, suggesting that the presence of a β‐phosphate is essential for significant binding of an ATP analog to the enzyme. Surprisingly, we found that inhibition by the well‐known p38 MAPKα inhibitor SB 203580 does not follow classical linear inhibition kinetics at concentrations > 100 n m , as previously suggested, demonstrating that caution must be used when interpreting kinetic experiments using this inhibitor.