Substrate analogue for the detection of specific prenylated methylated protein methyl esterase (PMPMEase) activity. Cholinesterases do not hydrolyse BzGFCM. roteins anchored to membranes via prenyl groups include yeast mating factors, the p21ras protein (the protein product of the ras oncogene), and the nuclear lamins, structural components of the lamina of the inner nuclear membrane
1 moreTitle: Insights into the physiological role of pig liver esterase: isoenzymes show differences in the demethylation of prenylated proteins Brusehaber E, Bottcher D, Bornscheuer UT Ref: Bioorganic & Medicinal Chemistry, 17:7878, 2009 : PubMed
The possible physiological role of PLE (E.C. 3.1.1.1) located in the endoplasmic reticulum (ER) of pig liver cells in the conversion of endogenous compounds was investigated as it was reported, that PLE acts as prenylated methylated protein methyl esterase (PMPMEase) hydrolysing methylesters of prenylated proteins. Using the specific PMPMEase substrate benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM), six different PLE isoenzymes expressed recombinantly in the yeast Pichia pastoris were found active. Activities ranged from 1.6-15.6mU per mg protein and it is suggested that Pro285 has a major influence on high activity. In addition, the role of the C-terminal HAEL retention signal for translocation of pig liver esterase (PLE) in the endoplasmic reticulum (ER) of eukaryotic cells was studied using the gamma-isoenzyme of PLE expressed in Pichia pastoris. Using truncated versions (HAE, HA, H and without retention signal) of the enzyme it was found that in contrast to earlier reports no influence of the signal peptide on the expression rate of PLE was found. However, higher enzyme activities were obtained in the periplasmatic fraction compared to the supernatant irrespective of the presence or absence of HAEL and the trimeric formation seems to occur in the supernatant of P. pastoris X33 enabling an easier transition of monomeric forms through cell membranes.
        
Title: Liver prenylated methylated protein methyl esterase is the same enzyme as Sus scrofa carboxylesterase Oboh OT, Lamango NS Ref: J Biochem Mol Toxicol, 22:51, 2008 : PubMed
The C-terminal --COOH of prenylated proteins is methylated to --COOCH3. The --COOCH3 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-L-cysteine methyl ester and hydrocinamoyl farnesyl-L-cysteine methyl ester with Michaelis-Menten constant (K(m)) values of 33 +/- 4 and 25 +/- 4 microM 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.
        
Title: Liver prenylated methylated protein methyl esterase is an organophosphate-sensitive enzyme Lamango NS Ref: J Biochem Mol Toxicol, 19:347, 2005 : PubMed
Prenylation and subsequent methylation are essential modifications on a significant proportion of eucaryotic proteins. Proteins such as the G-gamma subunits of G-protein coupled receptors, nuclear lamins, and guanine nucleotide-binding proteins such as Ras are prenylated and undergo methylation. Prenylated methylated protein methyl esterase (PMPMEase) readily hydrolyses the prenylated protein methyl esters, thus making this step reversible and possibly regulatory. Benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM) was developed as a specific PMPMEase substrate and characterized by electron spray ionization mass spectrometry (ESI-MS) to be of the calculated molecular mass. Rat liver and brain PMPMEase hydrolyzed BzGFCM, forming benzoyl-glycyl-farnesyl-cysteine (BzGFC) in a time- and concentration-dependent manner. Both enzymes cleaved BzGFCM with K(m) values of 4.58 +/- 0.30 and 25.57 +/- 2.36 microM and V(max) values of 2.21 +/- 0.03 and 0.17 +/- 0.003 nmol/min/mg, respectively. The liver enzyme eluted from a gel-filtration column as a single peak of apparent size, 89 kDa. The brain enzyme eluted as two main peaks of 53 and 890 kDa. Organophosphorus pesticides (OPs), which are suspected to be involved in human disorders such as parkinsonism, neuronal, and retinal degeneration, inhibited the liver enzyme with IC(50) values from 4.77 muM for parathion to 0.04 microM for paraoxon, respectively. Only about 25% of the brain enzyme was inhibited by 0.5-1 mM solutions of mipafox, while 0.1 and 1 mM paraoxon inhibited over 50% and 95% of the enzyme, respectively. Paraoxon is thus about 2,250 times less potent against the brain than the liver PMPMEase. BzGFCM was not hydrolyzed by various cholinesterases, indicating its specificity for PMPMEase. Perturbations in prenylated protein metabolism might play a role in noncholinergic OPs-induced toxicity, since prenylated proteins play such important roles in cell signaling, proliferation, differentiation, and apoptosis.
        
1 lessTitle: Insights into the physiological role of pig liver esterase: isoenzymes show differences in the demethylation of prenylated proteins Brusehaber E, Bottcher D, Bornscheuer UT Ref: Bioorganic & Medicinal Chemistry, 17:7878, 2009 : PubMed
The possible physiological role of PLE (E.C. 3.1.1.1) located in the endoplasmic reticulum (ER) of pig liver cells in the conversion of endogenous compounds was investigated as it was reported, that PLE acts as prenylated methylated protein methyl esterase (PMPMEase) hydrolysing methylesters of prenylated proteins. Using the specific PMPMEase substrate benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM), six different PLE isoenzymes expressed recombinantly in the yeast Pichia pastoris were found active. Activities ranged from 1.6-15.6mU per mg protein and it is suggested that Pro285 has a major influence on high activity. In addition, the role of the C-terminal HAEL retention signal for translocation of pig liver esterase (PLE) in the endoplasmic reticulum (ER) of eukaryotic cells was studied using the gamma-isoenzyme of PLE expressed in Pichia pastoris. Using truncated versions (HAE, HA, H and without retention signal) of the enzyme it was found that in contrast to earlier reports no influence of the signal peptide on the expression rate of PLE was found. However, higher enzyme activities were obtained in the periplasmatic fraction compared to the supernatant irrespective of the presence or absence of HAEL and the trimeric formation seems to occur in the supernatant of P. pastoris X33 enabling an easier transition of monomeric forms through cell membranes.
        
Title: Porcine Liver Carboxylesterase Requires Polyisoprenylation for High Affinity Binding to Cysteinyl Substrates Lamango NS, Duverna R, Zhang W, Ablordeppey SY Ref: Open Enzym Inhib J, 2:12, 2009 : PubMed
The polyisoprenylation pathway enzymes have been the focus of numerous studies to better understand the roles of polyisoprenylated proteins in eukaryotic cells and to identify novel targets against diseases such as cancer. The final step of the pathway is a reversible reaction catalyzed by isoprenyl carboxylmethyl transferase (icmt) whose products are then hydrolyzed by polyisoprenylated methylated protein methyl esterase (PMPMEase). Unlike the other pathway enzymes, the esterase has received little attention. We recently purified PMPMEase from porcine liver using an S-polyisoprenylated cysteine methyl ester substrate-dependent screening assay. However, no data is available showing its relative interaction with structurally diverse substrates. As such, its role as the putative endogenous PMPMEase has not been demonstrated. A series of substrates with S-alkyl substituents ranging from 2 to 20 carbons, including the two moieties found in polyisoprenylated proteins, were synthesized. Enzyme kinetics analysis revealed a 33-fold increase in affinity (K(M) values) from ethyl- (C-2, 505+/-63 microM), prenyl- (C-5, 294+/-25 microM), trans-geranyl- (C-10, 87+/-12 microM), trans, trans-farnesyl- (C-15, 29+/-2.2 microM) to all trans-geranylgeranyl- (C-20-, 15+/-2.7 microM) based analogs. Comparative molecular field analysis of the data yielded a cross-validated q(2) of 0.863+/-0.365 and a final R(2) of 0.995. Since the substrates with the S-trans, trans-farnesyl and S-all trans-geranylgeranyl moieties that occur in proteins show the highest affinity towards PMPMEase and are not hydrolyzed by the cholinesterases, the results suggest that polyisoprenylated proteins are the endogenous substrates of this esterase. The results suggest design strategies for high affinity and selective inhibitors of PMPMEase.
        
Title: Liver prenylated methylated protein methyl esterase is the same enzyme as Sus scrofa carboxylesterase Oboh OT, Lamango NS Ref: J Biochem Mol Toxicol, 22:51, 2008 : PubMed
The C-terminal --COOH of prenylated proteins is methylated to --COOCH3. The --COOCH3 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-L-cysteine methyl ester and hydrocinamoyl farnesyl-L-cysteine methyl ester with Michaelis-Menten constant (K(m)) values of 33 +/- 4 and 25 +/- 4 microM 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.
        
Title: Liver prenylated methylated protein methyl esterase is an organophosphate-sensitive enzyme Lamango NS Ref: J Biochem Mol Toxicol, 19:347, 2005 : PubMed
Prenylation and subsequent methylation are essential modifications on a significant proportion of eucaryotic proteins. Proteins such as the G-gamma subunits of G-protein coupled receptors, nuclear lamins, and guanine nucleotide-binding proteins such as Ras are prenylated and undergo methylation. Prenylated methylated protein methyl esterase (PMPMEase) readily hydrolyses the prenylated protein methyl esters, thus making this step reversible and possibly regulatory. Benzoyl-glycyl-farnesyl-cysteine methyl ester (BzGFCM) was developed as a specific PMPMEase substrate and characterized by electron spray ionization mass spectrometry (ESI-MS) to be of the calculated molecular mass. Rat liver and brain PMPMEase hydrolyzed BzGFCM, forming benzoyl-glycyl-farnesyl-cysteine (BzGFC) in a time- and concentration-dependent manner. Both enzymes cleaved BzGFCM with K(m) values of 4.58 +/- 0.30 and 25.57 +/- 2.36 microM and V(max) values of 2.21 +/- 0.03 and 0.17 +/- 0.003 nmol/min/mg, respectively. The liver enzyme eluted from a gel-filtration column as a single peak of apparent size, 89 kDa. The brain enzyme eluted as two main peaks of 53 and 890 kDa. Organophosphorus pesticides (OPs), which are suspected to be involved in human disorders such as parkinsonism, neuronal, and retinal degeneration, inhibited the liver enzyme with IC(50) values from 4.77 muM for parathion to 0.04 microM for paraoxon, respectively. Only about 25% of the brain enzyme was inhibited by 0.5-1 mM solutions of mipafox, while 0.1 and 1 mM paraoxon inhibited over 50% and 95% of the enzyme, respectively. Paraoxon is thus about 2,250 times less potent against the brain than the liver PMPMEase. BzGFCM was not hydrolyzed by various cholinesterases, indicating its specificity for PMPMEase. Perturbations in prenylated protein metabolism might play a role in noncholinergic OPs-induced toxicity, since prenylated proteins play such important roles in cell signaling, proliferation, differentiation, and apoptosis.