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The polyisoprenylation pathway incorporates a reversible step that metabolizes polyisoprenylated methylated proteins from the ester to the carboxylate form. Polyisoprenylated protein methyl transferase (PPMTase) catalyses the esterification whereas polyisoprenylated methylated protein methyl esterase (PMPMEase) hydrolyzes them. Significant changes in the balance between the two enzymes may alter polyisoprenylated protein function possibly resulting in disease. Previous studies show that PMPMEase is the serine hydrolase, Sus scrofa carboxylesterase. Its susceptibility to the nonspecific serine hydrolase inhibitor, phenylmethylsulfonyl fluoride (PMSF) paved the way for its use as a prototypical compound to design and synthesize a series of putative high affinity specific inhibitors of PMPMEase. Pseudo first-order kinetics revealed an over 680-fold increase in k(obs)/[I] values from PMSF (6 M(-1)-1s(-1)), S-phenyl (L-50, 180 M(-1)s(-1)), S-benzyl (L-51, 350 M(-1)s(-1)), S-trans, trans-farnesyl (L-28, 2000 M(-1)s(-1)), to S-trans-geranylated (L-23, 4100 M(-1)s(-1)) 2-thioethanesulfonyl fluorides. C10 S-alkyl substitution revealed a k(obs)/[I] value (1800 M(-1)s(-1)) that was 298 times greater than that for PMSF. The compounds induced the degeneration of human neuroblastoma SH-SY5Y cells with EC(50) values of 49, 130 and1000 µM for L-28, L-23 and PMSF, respectively. The increased affinity with the polyisoprenyl derivatization is consistent with the observed substrate specificity and the reported hydrophobic nature of the active site. These results suggest that (1) PMPMEase is a key enzyme for polyisoprenylated protein metabolism, (2) regulation of its activity is essential for maintaining normal cell viability, (3) abnormal activities may be involved in degenerative diseases and cancers and (4) its specific inhibitors may be useful in combating cancers.

Polyisoprenylated proteins (PPs) methylation by polyisoprenylated protein methyl transferase (PPMTase) is counteracted by polyisoprenylated methylated protein methyl esterase (PMPMEase). This is the only reversible step of the polyisoprenylation pathway as the relative amounts of the acid and ester forms are determined by the two competing reactions. Since PMPMEase and PPMTase may influence both the structural/functional conformations of PPs, a thorough study of these enzymes is essential to our understanding of the structural/functional features of PPs. PMPMEase has been reported under such pseudonyms as human carboxylesterase 1 (hCE1) because of its apparent broad substrate spectrum. The current study aimed to show the complementarity between its active site and the polyisoprenylated substrates that it metabolizes. Kinetics analysis was conducted with N-, S- and O-substituted substrates using porcine liver PMPMEase and docking analysis using Arguslab. Consistent with the biochemical analysis, the S-ethyl analog yielded an AScore binding energy of -11.32 compared to -13.48, -14.88, -16.15, and -16.81 kcal/mol for S-prenyl (C-5), S-trans-geranyl (C-10), S-trans,trans-farnesyl (C-15) and S-all trans-geranylgeranyl (C-20), respectively. The all trans-geranylgeranyl moiety provides the optimal size for active site interactions. The data reveal that the trans,trans-farnesyl and all trans-geranylgeranyl groups, which are reminiscent of endogenous PPs modifications, have the highest affinity for PMPMEase. Since PPs such as monomeric G-proteins are oncogenic, PMPMEase may be viewed as a viable target for anticancer drug development. The analyses reveal the important structural elements for the design of specific PMPMEase inhibitors to serve in the modulation of oncogenic PPs activities. The results also show that hCE1's repertoire of substrates extends beyond xenobiotics to include PPs as its endogenous substrates.