A Revised Mechanism for Human Cyclooxygenase-2.

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Cyclooxygenase 2 (COX-2) catalyzes the oxidation of polyunsaturated fatty acids.
• The precise initial steps and mechanism of COX-2 catalysis remain incompletely understood.
• A conserved hydrogen bond involving the catalytic tyrosyl radical/tyrosine is crucial for COX-2 function.

Purpose of the Study:

• To elucidate the initial irreversible step in ω-6 polyunsaturated fatty acid oxidation by wild-type COX-2 and its Y334F variant.
• To investigate the role of the conserved hydrogen bond in COX-2 catalysis.
• To differentiate between proposed catalytic mechanisms under physiologically relevant conditions.

Main Methods:

• Enzyme kinetics studies using wild-type and Y334F variant cyclooxygenase 2.
• Measurement of apparent bimolecular rate constants and deuterium kinetic isotope effects.
• Temperature-dependent kinetic analyses.

Main Results:

• Apparent bimolecular rate constants and deuterium kinetic isotope effects increased with co-substrate concentration, then reached limiting values.
• Observed kinetic trends excluded multiple dioxygenase mechanisms.
• Temperature-dependent studies identified hydrogen transfer from reduced catalytic tyrosine to a terminal peroxyl radical as the first irreversible step.

Conclusions:

• The initial irreversible step in COX-2 catalysis is hydrogen transfer from the catalytic tyrosine to a peroxyl radical, not initial hydrogen atom abstraction from the fatty acid.
• This finding clarifies the mechanism of regio- and stereospecific product formation by COX-2.
• The Y334F mutation's impact on catalysis provides insights into the role of the conserved hydrogen bond.