Kinetic mechanism and ATP-binding site reactivity of p38gamma MAP kinase.
Activated p38gamma MAP kinase exhibited significant basal ATPase activity in the absence of a kinase substrate, and addition of a phosphoacceptor substrate increased k(cat)/K(m)20-fold. AMP-PCP was competitive with ATP binding and non-competitive with phosphoacceptor substrate binding. The nucleotide binding site affinity label 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA) bound stoichiometrically at Lys-56 in the ATP site of both unphosphorylated and activated p38gamma. AMP-PCP only protected the activated enzyme from FSBA inactivation, implying that AMP-PCP does not bind unphosphorylated p38gamma. Basal ATPase activities were also observed for activated p38alpha, ERK2 and JNK3 suggesting that the enzymatic mechanism may be similar for all classes of MAP kinases.
Fox
T
T
Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, MA 02139-4242, USA. Fox@vpharm.com
Fitzgibbon
M J
MJ
Fleming
M A
MA
Hsiao
H M
HM
Brummel
C L
CL
Su
M S
MS
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels; D000495:Allosteric Site; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004789:Enzyme Activation; D007700:Kinetics; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048057:Mitogen-Activated Protein Kinase 10; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases
D000345:Affinity Labels; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D048057:Mitogen-Activated Protein Kinase 10; D011505:Protein-Tyrosine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000251:Adenosine Triphosphatases; D000241:Adenosine; C002630:alpha,beta-methyleneadenosine 5'-triphosphate