Porcine Liver Carboxylesterase Requires Polyisoprenylation for High Affinity Binding to Cysteinyl Substrates.
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.
Lamango
Nazarius S
NS
College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, FL 32307, USA.
Duverna
Randolph
R
Zhang
Wang
W
Ablordeppey
Seth Y
SY