Structure-function relationships in sheep, mouse, and human prostaglandin endoperoxide G/H synthases

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Prostaglandin endoperoxide (PGG/H) synthase catalyzes key steps in prostaglandin synthesis.
• Understanding the enzyme's active site is crucial for drug development targeting inflammation and pain.

Purpose of the Study:

• To identify critical amino acid residues responsible for the cyclooxygenase and hydroperoxidase activities of PGG/H synthase.
• To elucidate the mechanism by which aspirin and ibuprofen interact with the enzyme.

Main Methods:

• Deduction of amino acid sequences from complementary (c)DNAs of sheep, mouse, and human PGG/H synthases.
• Site-directed mutagenesis of Serine 530 to Alanine and Asparagine.
• Enzyme activity assays for cyclooxygenase and hydroperoxidase functions.
• Chemical inactivation studies using tetranitromethane and protection assays with ibuprofen.

Main Results:

• High sequence identity (~90%) observed between sheep, mouse, and human PGG/H synthases.
• Mutagenesis of Ser530 to Ala530 retained both enzyme activities, while Asn530 abolished cyclooxygenase activity.
• A bulky group at position 530, like aspirin acetylation, inhibits arachidonate binding.
• Tetranitromethane irreversibly inactivated cyclooxygenase activity, with ibuprofen providing protection, suggesting an essential tyrosine residue.

Conclusions:

• The hydroxyl group of Ser530 is not essential for PGG/H synthase catalysis but its acetylation by aspirin blocks substrate access.
• A specific tyrosine residue in the active site is essential for cyclooxygenase activity.
• These findings provide insights into the mechanism of action for NSAIDs like aspirin and ibuprofen.