Kinetic mechanism of the p38-alpha MAP kinase: Phosphoryl transfer to synthetic peptides

p38 is a member of the mitogen-activated protein (MAP) kinase family. Activ ation (phosphorylation) of p38 acts as a switch for the transcriptional and translational regulation of a number of proteins, including the proinflamm atory cytokines. Investigation of a set of small peptides revealed that, as with protein substrates, p38-alpha behaves as a proline-directed Ser/Thr M AP kinase for a peptide substrate, peptide 4 (IPTSPITTTYFFFKKK). We investi gated the steady-state kinetic mechanism of the p38-alpha-catalyzed kinase reaction with EGF receptor peptide, peptide 1, as a substrate. Lineweaver-B urk analysis of the substrate kinetics yielded a family of lines intersecti ng to the left of the ordinate, with either ATP or peptide 1 as the varied substrate. Kinetic analysis in the presence of ADP yielded a competitive in hibition pattern when ATP was the varied substrate and a noncompetitive pat tern if peptide 1 was the varied substrate. At saturating peptide substrate concentrations, inhibition by phosphopeptide product yielded an uncompetit ive pattern when ATP was the varied substrate. These data are consistent wi th ordered binding with ATP as the initial substrate. We provide further ev idence of the existence of a productive p38 ATP binary complex in that (a) activated p38-alpha has intrinsic ATPase activity, (b) ATPase and kinase ac tivities are coupled, and (c) inhibitors of ATPase activity also inhibit th e kinase activity with a similar inhibition constant. The k(cat) for the ki nase reaction was lowered by 1.8-fold when ATP-gamma-S was used. Microvisco sity linearly affected the k(cat) values of both the ATP and ATP-gamma-S re actions with a slope of about 0.8. These observations were interpreted to m ean that the phosphoryl transfer step is not rate-limiting and that the rel ease of product and/or enzyme isomerization is a possible rate-limiting ste p(s).