Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.16.2 (PCP)
 3,761 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Kinetic mechanism for p38 MAP kinase. 926 22

To study interactions between the contiguous NBD1 and R domains of CFTR, wild-type and DeltaF508 NBD1-R (amino acids 404-830, in fusion with His6 tag) were expressed as single proteins in Escherichia coli. NBD1-R (10-25 mg/L culture) was purified from inclusion bodies in 8 M urea by Ni-affinity chromatography, and renatured by rapid dilution at pH 5. In vitro phosphorylation by protein kinase A increased the apparent size of NBD1-R from approximately 52 to approximately 56 kDa by SDS-PAGE. The fluorescent ATP analogue TNP-ATP bound to renatured NBD1-R with of 0.81 +/- 0.1 microM (wild-type), 0.93 +/- 0.1 microM (wild-type, phosphorylated), 0.75 +/- 0.1 microM (DeltaF508 NBD1-R), and 0.72 +/- 0.1 microM (DeltaF508 NBD1-R, phosphorylated) with a stoichiometry of approximately 1 TNP-ATP site per NBD1-R molecule; TNP-ATP binding was reversed by ATP, AMP-PCP, and AMP-PNP with KIs of approximately 3.2, 4.2, and 4.6 mM, respectively. Secondary structure analysis by circular dichroism gave 19% alpha-helix, 43% beta-sheet and turn, and 38% "other" structure. To determine if nucleotide binding to NBD1 influenced R domain phosphorylation, NBD1-R was in vitro phosphorylated with protein kinase A and [gamma-32P]ATP in the presence of AMP-PCP, AMP-PNP, or TNP-ATP. Whereas the nucleotide analogues did not affect 32P-incorporation in control proteins (Kemptide, GST-R domain), phosphorylation of NBD1-R was reduced >75% by AMP-PNP or AMP-PCP (0.25 mM) and >50% by TNP-ATP (0.25 microM). Analysis of phosphorylation sites indicated that inhibition involved multiple sites in NBD1-R, including serines 660, 712, 737, 795, and 813. These results establish the conditions for NBD1-R expression, purification, and renaturation. The inhibition of R domain phosphorylation by nucleotide binding to the NBD1 domain indicates significant domain-domain interactions and suggests a novel mechanism for regulation of CFTR phosphorylation.
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PMID:Expression and characterization of the NBD1-R domain region of CFTR: evidence for subunit-subunit interactions. 948 88