Back to Search
Start Over
Involvement of Arginine 120, Glutamate 524, and Tyrosine 355 in the Binding of Arachidonate and 2-Phenylpropionic Acid Inhibitors to the Cyclooxygenase Active Site of Ovine Prostaglandin Endoperoxide H Synthase-1
- Source :
- Journal of Biological Chemistry. 271:2179-2184
- Publication Year :
- 1996
- Publisher :
- Elsevier BV, 1996.
-
Abstract
- Examination of the crystal structure of the ovine prostaglandin endoperoxide synthase-1 (PGHS-1)/S- flurbiprofen complex (Picot, D., Loll, P.J., and Garavito, R.M. (1994) Nature 367, 243-2491) suggests (a) that the carboxyl group of arachidonic acid interacts with the arginino group of Arg120; (b) that Arg120 forms an important salt bridge with Glu524; and (c) that Tyr355, which is in close proximity to Arg120, could determine the stereochemical specificity of PGHS-1 toward 2-phenylpropionic acid inhibitors. To test these concepts, we used site-directed mutagenesis to prepare ovine PGHS-1 mutants having modifications of Arg120 (R120K, R120Q, R120E), Glu524 (E524D, E524Q, E524K), and Tyr355 (Y355F) and examined the properties of the mutant enzymes expressed in COS-1 cells. All of the mutants retained at least part of their cyclooxygenase and peroxidase activities except the R120E mutant, which had no detectable activity. The Km values of the R120K and R120Q mutants with arachidonic acid were 87 and 3300 microM, respectively, versus 4 microM for native PGHS-1. The R120Q mutant failed to undergo suicide inactivation during catalysis or time-dependent inhibition by flurbiprofen. These results are consistent with Arg120 binding the carboxylate group of arachidonate and suggest that interaction of the carboxylate group of substrates and inhibitors with Arg120 is necessary for suicide inactivation and time-dependent inhibition, respectively. The Km values for the E524D, E524Q, and E524K mutants were not significantly different from values obtained for the native PGHS-1, suggesting that this residue is not importantly involved in catalysis or substrate binding. The effect of modifications of Arg120 and Tyr355 on the stereospecificity of inhibitor binding was determined. Ratios of IC50 values for cyclooxygenase inhibition by D- and L-ibuprofen, a competitive cyclooxygenase inhibitor, were 32, 67, and 7.1 for native PGHS-1, R120Q PGHS-1, and Y355F PGHS-1, respectively. The decreased stereochemical specificity observed with the Y355F PGHS-1 mutant suggests that Tyr355 is a determinant of the stereospecificity of PGHS-1 toward inhibitors of the 2-phenylpropionic acid class.
- Subjects :
- Models, Molecular
Arginine
Stereochemistry
Molecular Sequence Data
Mutant
Ibuprofen
Biochemistry
Structure-Activity Relationship
chemistry.chemical_compound
Glutamates
Animals
Cyclooxygenase Inhibitors
Amino Acid Sequence
Enzyme Inhibitors
Tyrosine
Binding site
Molecular Biology
DNA Primers
chemistry.chemical_classification
Arachidonic Acid
Binding Sites
Sheep
Base Sequence
Phenylpropionates
biology
Chemistry
Active site
Cell Biology
Protein Structure, Tertiary
Kinetics
Enzyme
Flurbiprofen
Prostaglandin-Endoperoxide Synthases
Mutagenesis, Site-Directed
biology.protein
Arachidonic acid
Cyclooxygenase
Subjects
Details
- ISSN :
- 00219258
- Volume :
- 271
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry
- Accession number :
- edsair.doi.dedup.....634084b055adf7eba4888c6c27b36f3e