Biochemical and Docking Analysis of Substrate Interactions with Polyisoprenylated Methylated Protein Methyl Esterase

Abstract

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 Sethyl 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 Sall 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 transgeranylgeranyl 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.

Polyisoprenylated methylated protein methyl esterase: A putative biomarker and therapeutic target for pancreatic cancer

“…Nonlinear regression analysis of the inhibitor-response curves were calculated using GraphPad Prism version 5.0 for Mac (San Diego, CA) and the concentrations that inhibit 50% of enzymatic activity (IC 50 ) was determined. The inhibition constant ( K i ) was then calculated from the previously reported kinetics analysis of RD-PNB substrate [7, 8]. …”

“…Docking analysis of the PCAIs was conducted as previously described [8, 9] with an increased potential binding area of 20×20×24 points. Poses were selected based on the interaction energy and the plausibility of the interactions between the PCAIs and the active site residues.…”

“…Polyisoprenylated protein methyl transferase (PPMTase) esterifies the carboxyl terminus whereas polyisoprenylated methylated protein methyl esterase (PMPMEase) hydrolyzes the ester bond. Substrate analysis of PMPMEase [7, 8] led to the synthesis of high affinity, irreversible sulfonyl fluoride inhibitors (Figure 1) [9]. Meanwhile, polyunsaturated fatty acids (PUFAs), which are known to exhibit anticancer activity, have been shown to inhibit PMPMEase [10].…”

Polyisoprenylated Methylated Protein Methyl Esterase Is Both Sensitive to Curcumin and Overexpressed in Colorectal Cancer: Implications for Chemoprevention and Treatment

“…Michaelis-Menten kinetics analysis was conducted using RD-PNB as the substrate as previously described [38]. Varying concentrations of the substrate (0–400  μ M) were incubated with PMPMEase (5  μ g) in the presence of curcumin (0-1 mM).…”

“…They were then placed on ice for at least 5 min before centrifugation at 5000 ×g for 5 min. The supernatants were removed and analyzed by RP-HPLC with UV detection at 260 nm as previously described [38]. The product peak areas were measured and used to quantify the amount of product formed using a calibration plot of known amounts of product against peak area.…”