Liver prenylated methylated protein methyl esterase is an organophosphate-sensitive enzyme

Abstract

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.

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

“…It shows high-sequence identity and even higher sequence similarity to various mammalian esterases that hydrolyze a wide range of mainly hydrophobic substrates and have molecular weights around 60 kDa [21][22][23]. These features are similar to those of the purified PMPMEase, which shows a molecular weight on SDS-PAGE of about 57 kDa, and both the rat liver [10] and porcine liver in this study show a high affinity for the hydrophobic PC analog ester substrates. The crystal structure of the human isoform, hCE1, reveals complexes of homotrimers at equilibrium with the homohexamers and the catalytic triad of Ser221-His468-Glu354 inside a deep hydrophobic pocket that has a small rigid subsite and a large flexible region [24,25].…”

“…Organophosphorus pesticides (OPs) [11][12][13], lactones [14], sulfonyl fluorides [15], and chloromethylketones [16] inhibit various serine hydrolases by this mechanism. In previous studies, prenylated methylated protein methyl esterase from rat liver supernatant and brain membranes was inhibited by OPs [10]. Similarly, a prenylation specific esterase from bovine rod outer segment membranes was inhibited by ebelactone B [14].…”

“…This functional specificity may be a product of coevolution that could have extended to include interactions of the prenylated proteins and PC analogs to PMPMEase. Rat liver supernatant and brain membranes containing PMPMEase were previously found to hydrolyze prenylated substrates with Michaelis-Menten constants of 5 and 25 µM, respectively [10]. Using such prenylated cysteine ester substrates to screen chromatographic fractions, it would be possible to selectively enrich endogenous esterases that metabolize prenylated proteins.…”

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

“…This substance was used as substrate analogue for the detection of specific PMPMEase activity, because various choline esterases did not hydrolyse BzGFCM. 13 The investigations were performed using PLE isoenzymes 1-6 (with cPLE being identical to PLE-1), commercially available PLE from Fluka and APLE. 18 Conversion was determined at different times using RP-HPLC.…”

“…[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.…”