[Prostaglandin H synthetase as a multisubstrate enzyme. Fluorimetric study of enzyme kinetics]

The kinetics of a multisubstrate enzymatic reaction catalyzed by prostaglandin H synthase (PGH-synthase, EC 1.14.99.1) was studied, using homovanillic acid, a new electron donor for the given system. Homovanillic acid was shown to be a participant in a reaction with arachidonic acid/O2 stoichiometric ratios and is oxidized to a readily fluorescing product with an absorbance maximum (excitation) at 315 nm and fluorescence maximum at 425 nm. This allows for determination of the rate of enzymatic reaction with the sensitivity exceeding by one order of magnitude that of polarographic or spectrophotometric assays. Using fluorescent techniques, the dependence of the rate of PGH-synthase reaction on substrate (arachidonic acid, O2 and homovanillic acid) concentrations was studied, and the corresponding Km values were determined. The effect of Tween-20 and Lubrol PX concentrations on the reaction rate were examined. It was shown that with a decrease in the surfactant concentration the reaction rate increases.