Development of a Coupled Enzyme Assay Method for Microsomal Prostaglandin E Synthase Activity

Abstract

Human microsomal prostaglandin E synthase-1 (mPGES-1) catalyzes the conversion of prostaglandin <TEX>$H_2$</TEX> (<TEX>$PGH_2$</TEX>) into prostaglandin <TEX>$E_2$</TEX> (<TEX>$PGE_2$</TEX>). To establish a stable and efficient method to assess the activity of mPGES-1, a coupled enzyme assay system using mPGES-1, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and phosphomolybdic acid (PMA) was developed. In this assay system, <TEX>$PGH_2$</TEX> was converted to <TEX>$PGE_2$</TEX> by mPGES-1, and then <TEX>$PGE_2$</TEX> was further transformed to the 15-keto-<TEX>$PGE_2$</TEX> by 15-PGDH accompanying the production of NADH, which was easily detected by fluorescence spectrometry in a multi-well plate format. During the reaction, spontaneous oxidation of <TEX>$PGH_2$</TEX> was prevented by PMA. Using this novel assay, the <TEX>$K_m$</TEX> value of mPGES-1 for <TEX>$PGH_2$</TEX> and the <TEX>$IC_{50}$</TEX> value of the previously characterized inhibitor, MK-886, were determined to be 0.150 mM and <TEX>$2.8\;{\mu}M$</TEX>, respectively, which were consistent with the previously reported values. In addition, low backgrounds were observed in the multi-wall plate screening of chemical compounds.