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 fro m 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 acc ordance with the claim that a functional serine residue is necessary f or the lipolytic activity of lipases. PGE was not inhibited by the thi ol 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-este rified fatty acid C-12:0. PGE shows a great sequence similarity to gas tric 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 function al free thiol group. The PGE cDNA codes only for two cysteines, and th eir involvement in the formation of a disulphide bridge was demonstrat ed.