Kinetic mechanism for p38 MAP kinase.
p38 has been shown to be a critical enzyme in the pro-inflammatory cytokine pathway and is a member of the mitogen-activated protein (MAP) kinase family. While the details for p38 activation and subsequent signal transduction have begun to be elucidated, little is known about the kinetic mechanism for p38. In this study, we have determined the kinetic mechanism for p38 MAP kinase. Data from initial velocity patterns in the presence and absence of a dead-end inhibitor and two triarylimidazole p38 inhibitors were consistent with an ordered sequential mechanism for p38 with protein substrate, glutathione S-transferase-activating transcription factor 2 (GST-ATF2), binding before ATP. The ATP analog, adenylyl methylenediphosphonate (AMP-PCP), and two triarylimidazoles were competitive inhibitors versus ATP and uncompetitive inhibitors versus GST-ATF2. Equilibrium binding studies utilizing a tritiated ATP-competitive inhibitor were also consistent with this mechanism and suggest an inability of ATP to bind to p38 in the absence of protein substrate. Moreover, the Michaelis constant for GST-ATF2 was 12-fold greater than the dissociation constant, indicating that the binding of ATP affected the binding of GST-ATF2. An ordered sequential mechanism with protein substrate binding first is unique to p38 compared to cyclic AMP-dependent protein kinase (cAPK) and most tyrosine kinases and helps to explain the interaction between enzyme, substrates, and inhibitors.
LoGrasso
P V
PV
Department of Molecular Design and Diversity, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, USA. lograsso@merck.com
Frantz
B
B
Rolando
A M
AM
O'Keefe
S J
SJ
Hermes
J D
JD
O'Neill
E A
EA
D051698:Activating Transcription Factor 2; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases; D015394:Molecular Structure; D010766:Phosphorylation; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000201:enzymology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D004791:Enzyme Inhibitors; D007093:Imidazoles; D011725:Pyridines; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; C093642:SB 203580; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole