Stereospecific and Regioselective Hydrolysis of Cannabinoid Esters by ES46.5K, an Esterase from Mouse Hepatic Microsomes, and Its Differences from Carboxylesterases of Rabbit and Porcine Liver

Abstract

Carboxylesterases are a family of enzymes that catalyze the hydrolysis of esters and amides. [1][2][3][4] Carboxylesterases have an important role in the detoxication of xenobiotics and in metabolic activation of prodrugs in some cases. [5][6][7][8] Multiple carboxylesterase isozymes have been purified from various animal species 3,4) including rabbits 9,10) and pigs. 11,12) Although the substrate specificities for many forms of purified carboxylesterases have been well characterized, little is known about their stereospecificity and regioselectivity for the hydrolysis of xenobiotic esters. In relation to the stereospecific hydrolysis of substrates by esterases, enzymatic resolution of enantiomers to obtain a useful chiral starting material has been reported using pig pancreatic lipase. 13,14) Gatley 15) reported that the unusual cocaine enantiomer, (ϩ)-cocaine, was hydrolyzed by butyrylcholine esterase over 2000 times faster than (Ϫ)-cocaine. Maksay et al. 16) reported that esterases in liver and brain homogenates of mice have opposite stereoselectivity in the hydrolysis of the racemate of oxazepam 3-acetate; the hepatic enzyme preferentially hydrolyzes the (R)-enantiomer, whereas the brain enzyme preferentially hydrolyzes the (S)-enantiomer. Yang and Lu 17) also reported that esterases in hepatic microsomes and brain homogenate of rats have the same opposite stereoselectivity in catalyzing the hydrolysis of enantiomeric oxazepam 3-acetate. The catalytic triad Ser/Asp/His is suggested to be a part of oxyanion hole as a catalytic site of various esterases and important for the hydrolysis of substrate esters by the enzymes.18) In addition, structural constraints also affect the interaction of substrates with carboxylesterases. 19) We previously purified the esterase ES46.5K from mouse hepatic microsomes and its role in the metabolism of xenobiotic esters has been characterized. [20][21][22] The enzyme is a 46.5-kDa esterase with considerable homology of the N-terminal amino acid sequence to arylacetamide deacetylase purified from human liver. 23) Recently, the enzyme has also been characterized as a microsomal lipase with arylamide deacetylase activity.24) The mammalian carboxylesterases represent a multigene family, the gene products of which are classified as CES1 to CES4. ES46.5K probably belongs to the CES4 family, as is the case for human acetamide deacetylase. 4)The present study describes the stereospecific and regioselective hydrolysis of cannabinoid esters as model substrates for ES46.5K and carboxylesterases from rabbit and porcine liver. In addition, differences in the characteristics between ES46.5K and the carboxylesterases were also investigated. The properties of ES46.5K, an esterase from mouse hepatic microsomes, were compared with those of carboxylesterases from rabbit and porcine liver. The inhibitory profile with a serine hydrolase inhibitor (bis-p-nitrophenylphosphate) and detergents (sodium dodecylsulfate, Emulgen 911) was different between ES46.5K and the carboxylesterases. Bis MATERIALS ...

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