Liver prenylated methylated protein methyl esterase is the same enzyme as sus scrofa carboxylesterase

Abstract

The C-terminal -COOH of prenylated proteins is methylated to -COOCH 3 . The -COOCH 3 ester forms are hydrolyzed by prenylated methylated protein methyl esterase (PMPMEase) to the original acid forms. This is the only reversible step of the prenylation pathway. PMPMEase has not been purified and identified and is therefore understudied. Using a prenylated-L-cysteine methyl ester as substrate, PMPMEase was purified to apparent homogeneity from porcine liver supernatant. SDS-PAGE analysis revealed an apparent mass of 57 kDa. Proteomics analyses identified 17 peptides (242 amino acids). A Mascot database search revealed these as portions of the Sus scrofa carboxylesterase, a 62-kDa serine hydrolase with the C-terminal HAEL endoplasmic reticulum-retention signal. It is at least 71% identical to such mammalian carboxylesterases as human carboxylesterase 1 with affinities toward hydrophobic substrates and known to activate prodrugs, metabolize active drugs, as well as detoxify various substances such as cocaine and food-derived esters. The purified enzyme hydrolyzed benzoyl-Gly-farnesyl-Lcysteine methyl ester and hydrocinamoyl farnesyl-L-cysteine methyl ester with Michaelis-Menten constant (K m ) values of 33 ± 4 and 25 ± 4 µM and V max values of 4.51 ± 0.28 and 6.80 ± 0.51 nmol/min/mg of protein, respectively. It was inhibited by organophosphates, chloromethyl ketones, ebelactone A and B, and phenylmethylsulfonyl fluoride.

Control of RhoA Methylation by Carboxylesterase I

“…Our approach to identifying a specific esterase responsible for the demethylation of RhoA observed in the MDA-MB-231 cells involved database analysis for proteins containing the esterase motif and examining the transcript expression profile of potential candidates, particularly targeting those expressed in mammary tissue and breast cancer cells. These criteria, along with homology to the recently identified porcine carboxylesterase capable of hydrolyzing prenylated cysteine methyl esters (20), led us to identify the CES family of esterases, and in particular the CES1 isoform. In parallel with assessment of CES1, we also examined the next best studied member of this family, CES2, which is more highly expressed in most tissues than CES1.…”

Insights into the physiological role of pig liver esterase: Isoenzymes show differences in the demethylation of prenylated proteins

“…12 They obtained esterase activity of 4.51 mU/mg protein towards BzGFCM from purified enzyme from pig liver containing an undefined composition of isoenzymes similar to commercial PLE from Fluka, which is a crude extract. This activity corresponds to the average of the PLE isoenzyme activity.…”

“…12 To elucidate the possible physiological role of the PLE isoenzymes discovered earlier in our group, these were expressed together with the chaperones GroEL/ES in E. coli Origami. After cell disruption and lyophilisation, the enzymes were used for the conversion of benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM).…”

“…[9][10][11] Oboh and Lamango purified the PMPMEase from pig liver and identified the 57 kDa protein as Sus scrofa carboxylesterase (pig liver esterase) after proteome analysis and Mascot database search. 12 The specific PMPMEase substrate, benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM) was synthesized 13 and it could be shown that this can be hydrolysed by the purified PMPMEase from pig liver. 12 As our group has succeeded in the past to identify, clone and functionally express several isoenzymes 14,15 of PLE, we report here also on the activity of the distinct isoenzymes in the hydrolysis of BzGFCM.…”

“…12 The specific PMPMEase substrate, benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM) was synthesized 13 and it could be shown that this can be hydrolysed by the purified PMPMEase from pig liver. 12 As our group has succeeded in the past to identify, clone and functionally express several isoenzymes 14,15 of PLE, we report here also on the activity of the distinct isoenzymes in the hydrolysis of BzGFCM.…”