Inhibition studies on calf pregastric esterase: the enzyme has no functional thiol group.
Pregastric esterase (PGE) (EC 3.1.1.3) was purified to homogeneity from calf pharyngeal tissue. The enzyme had an apparent molecular mass of 50 kDa, as determined by SDS/PAGE. The serine-binding reagent diethyl p-nitrophenyl phosphate was a potent inhibitor of PGE. This is in accordance with the claim that a functional serine residue is necessary for the lipolytic activity of lipases. PGE was not inhibited by the thiol reagents 5,5'-dithiobis(2-nitrobenzoic acid) or 4,4'-dithiopyridine. A partial inhibition with dodecyldithio-5-(2-nitrobenzoic acid) was observed, but the same degree of inhibition was caused by the non-esterified fatty acid C(12:0). PGE shows a great sequence similarity to gastric lipases. Gastric lipases have three cysteine residues, and two of these form a disulphide bridge. Blocking the remaining free cysteine with thiol reagents inactivates the gastric lipases. The fact that PGE is not inhibited by thiol reagents indicates that PGE has no functional free thiol group. The PGE cDNA codes only for two cysteines, and their involvement in the formation of a disulphide bridge was demonstrated.
Timmermans
M Y
MY
Department of Biochemistry, Limburgs Universitair Centrum, Diepenbeek, Belgium.
Reekmans
G
G
Teuchy
H J
HJ
Kupers
L P
LP
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010261:Paraoxon / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010614:Pharynx / Q000201:enzymology*; D011725:Pyridines / Q000494:pharmacology; D012694:Serine
D004220:Disulfides; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D011725:Pyridines; C007542:4,4'-dipyridyl disulfide; D012694:Serine; D004228:Dithionitrobenzoic Acid; D008049:Lipase; C043101:gastric triacylglycerol lipase; D010261:Paraoxon