Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2.
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.
Schindler
John F
JF
Signal Transduction and Enzymology Group, Pharmacia Corporation, Chesterfield Parkway North, St. Louis, MO 63198, USA. John.F.Schindler@Pharmacia.com
Godbey
Andrew
A
Hood
William F
WF
Bolten
Suzanne L
SL
Broadus
Richard M
RM
Kasten
Thomas P
TP
Cassely
Aaron J
AJ
Hirsch
Jeffery L
JL
Merwood
Michelle A
MA
Nagy
Mark A
MA
Fok
Kam F
KF
Saabye
Matthew J
MJ
Morgan
Heidi M
HM
Compton
Robert P
RP
Mourey
Robert J
RJ
Wittwer
Arthur J
AJ
Monahan
Joseph B
JB
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D000099190:MAP-Kinase-Activated Kinase 2
D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D000099190:MAP-Kinase-Activated Kinase 2