Alkylaryl sulfides as Peroxidase Reducing Substrates for Prostaglandin H Synthase: Probes for the Reactivity and Environment of the Ferryl-Oxo Complex

Abstract

Abstract The peroxidase activity of prostaglandin (PGH) synthase catalyzes the reduction of PGG2 and other natural and synthetic hydroperoxides by reducing substrates. Sulfides serve as reductants by incorporating the oxo ligand from the ferryl-oxo complex which represents the higher oxidation state of the peroxidase (Compound I). A series of alkylaryl sulfides and substituted dihydrobenzo[b]thiophenes were synthesized to determine the electronic and steric requirements of PGH synthase for sulfide reducing substrates. Kinetic parameters were determined for most of the molecules by determining their ability to support reduction of 5-phenyl-4-pentenyl-1-hydroperoxide in the presence of PGH synthase purified from ram seminal vesicle microsomes. Electron-donating groups on the aryl moiety para to the sulfide enhanced reducing substrate activity (p = -0.8). As expected from previous results, the major oxidation product of p-methylthioanisole was the corresponding sulfoxide. The presence of a para-amino group increased binding to the enzyme and changed the reduction mechanism from oxygen transfer to electron transfer. The major oxidation product of p-(dimethylamino)thioanisole was identified as p-(methylamino)thioanisole; an equivalent amount of formaldehyde was produced. Increasing the size of the alkyl group attached to sulfur decreased the ability of the sulfide to act as a peroxidase reductant. The maximal turnover for reduction by p-methoxyphenylalkyl sulfides decreased 10-fold on substitution of isopropyl for ethyl. Chiral derivatives of benzo[b]thiophenes demonstrated differences in the ability of the two enantiomers to support reduction. Introduction of a carboxylic acid moiety anywhere in the molecule decreased the maximal turnover for reduction. Esterification of the carboxylate doubled the extent of reduction relative to the free acid. The results are used to develop models for the interaction of sulfides with Compound I of PGH synthase.