Prostaglandin H synthase: resolved and unresolved mechanistic issues

Area of Science:

• Biochemistry
• Enzymology
• Chemical Kinetics

Background:

• Prostaglandin H synthase (PGHS)-1 and -2 possess intricate cyclooxygenase and peroxidase activities.
• These enzymes display complex kinetics, including feedback activation by peroxide and self-inactivation processes for both catalytic functions.

Purpose of the Study:

• To elucidate the mechanistic basis for the complex, non-linear steady-state kinetics of PGHS-1 and -2.
• To detail the branched chain radical mechanism governing PGHS catalysis.

Main Methods:

• Structure/function analyses
• Spectroscopic techniques
• Transient kinetic analyses
• Oxygen isotope effect studies
• Electron paramagnetic resonance (EPR) spectroscopy
• Radical trapping experiments

Main Results:

• PGHS catalysis is explained by a branched chain radical mechanism involving heme-based peroxidase and radical-based cyclooxygenase cycles.
• The Tyr385 radical acts as a crucial link, initiating the cyclooxygenase cycle by abstracting hydrogen from the fatty acid substrate.
• Peroxidase self-inactivation is linked to a side reaction involving the oxyferryl heme oxidant and the Tyr385 radical.
• Arachidonate radical formation is reversible, supported by spectroscopic and thermodynamic data.
• An alternate Tyr504 radical, potentially a reservoir of oxidizing equivalents, is linked to cyclooxygenase activation.

Conclusions:

• The interplay of radicals, particularly Tyr385 and Tyr504, and their interconversions are central to the regulation and inactivation of PGHS isozymes.
• Further investigation is ongoing to fully understand these radical dynamics in both PGHS-1 and PGHS-2.