Inhibition studies on calf pregastric esterase: the enzyme has no functional thiol group

Abstract

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.

Cysteine residues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity

“…In fact, the substitution of cysteines with residues such as alanine or phenylalanine does not result in a completely inactive enzyme. It is interesting to note that a similar absence of functional thiol groups has been observed recently in the calf pregastric esterase [23], a member of the acid lipase family, whereas dog, rabbit and human gastric lipases have been shown to be thiol enzymes [21,22].…”

“…Gastric lipase has been shown to be a thiol enzyme on the basis of chemical modification by thiol-reactive agents [21,22]. In contrast, the calf pregastric esterase has been reported to contain no functional thiol group [23]. Using a baculovirus expression system, Ser-153 has been shown to be important for the catalytic activity of LAL, even if it is not the actual catalytic nucleophile [20].…”

Purification of pregastric lipases of caprine origin

“…Purified forms of the PGL are less stable (4,7,8) and easily denatured at a lipid/water interface. Addition of BSA (up to 25 µM) stabilized GPGL (Fig.…”

“…Early investigations on the pregastric enzymes have been based on the crude or partially purified enzymes (3), and the structure and mechanism of their catalyzed reactions have not been discussed. Recently, however, the purification of the lin-gual lipases or PGL from calf (4)(5)(6), lamb (7,8), rat (9,10), and human (10)(11)(12) has been achieved, and the lipases reveal similar characteristics, even when extracted from different animals. Determination of the molecular weight of these PGL shows them to be glycoproteins with a high ratio of carbohydrate content and with molecular weights in the range of 43-51 kDa and highly homologous amino acid sequences.…”