@article {Wang1329, author = {Jie Wang and Eric T. Williams and Joanne Bourgea and Y. Nancy Wong and Christopher J. Patten}, title = {Characterization of Recombinant Human Carboxylesterases: Fluorescein Diacetate as a Probe Substrate for Human Carboxylesterase 2}, volume = {39}, number = {8}, pages = {1329--1333}, year = {2011}, doi = {10.1124/dmd.111.039628}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Human carboxylesterase (CES) 1 and CES2 are members of the serine hydrolase superfamily, and both exhibit broad substrate specificity and are involved in xenobiotic and endobiotic metabolism. Although expression of CES1 and CES2 occurs in several organs, their expression in liver and small intestine is predominantly attributed to CES1 and CES2, respectively. We successfully expressed CES1 form b (CES1-b) and form c (CES1-c) as well as CES2 in baculovirus-infected High Five insect cells. With 4-nitrophenyl acetate (4-NPA) as the probe substrate, the Km values of recombinant CES1-b and CES2 matched those of human liver microsomes (HLM) and human intestinal microsomes (HIM) with approximately 200 and 180 μM, respectively. Bis(4-nitrophenyl) phosphate potently inhibited 4-NPA hydrolysis by HLM, CES1-b, CES1-c, HIM, and CES2 with IC50 values less than 1 μM. With fluorescein diacetate (FD) as the substrate, the Km values were similar for all enzyme systems, with the exception of CES1-b, which was slightly lower; however, the Vmax values for HIM and CES2 were 39.5 and 14.6 μmol {\textperiodcentered} mg-1 {\textperiodcentered} min-1, respectively, which were at least 50-fold higher than those of CES1-b or CES1-c. Loperamide potently inhibited HLM, HIM, and CES2 with similar IC50 values of approximately 1 μM. Substrate specificity was compared between human tissues and recombinant enzymes. The data suggest the following: 1) FD is a probe substrate for CES2; 2) CES1-b is the predominant form in human liver; and 3) recombinant CES1-b and CES2 expressed in insect cells are functionally consistent with native carboxylesterases expressed in human liver and intestine, respectively.}, issn = {0090-9556}, URL = {https://dmd.aspetjournals.org/content/39/8/1329}, eprint = {https://dmd.aspetjournals.org/content/39/8/1329.full.pdf}, journal = {Drug Metabolism and Disposition} }