Insights into the physiological role of pig liver esterase: isoenzymes show differences in the demethylation of prenylated proteins.
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.
Brüsehaber
Elke
E
Institute of Biochemistry, Department of Biotechnology, Greifswald University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany.
Böttcher
Dominique
D
Bornscheuer
Uwe T
UT
D020816:Amino Acid Motifs; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004950:Esterases / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008745:Methylation; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D004950:Esterases