Hydrolysis of <i>p</i>‐nitrophenyl acetate by the peptide chain fragment (336–449) of porcine pancreatic lipase

Abstract

The incubation of porcine pancreatic lipase (449 amino acids) with chymotrypsin led to the preferential cleavage of the Phe-335-Ala-336 bond [Bousset-Risso et al. (1985) FEBS Lett. 182, 323-326]. Up to now it has not been possible to isolate the fragment (1-335) whereas fragment (336-449) was purified. This fragment does not display any activity towards the specific substrates of lipase, triacylglycerols, either in the aggregate form or monomeric solution, but like lipase it hydrolyzes p-nitrophenyl acetate. The biphasic kinetics of the release of p-nitrophenol by the fragment with different concentrations of p-nitrophenyl acetate ([S] greater than [E]) are very similar to those of lipase and other esterases. The initial burst is equal to 1 mol p-nitrophenol/mol fragment (when [S] = infinity). Ethoxyformic anhydride only reacts with 1 mol histidine out of the 2 mol that the fragment contained. The activity of the fragment towards p-nitrophenyl acetate hydrolysis is inhibited after ethoxyformic anhydride reaction as in the case of lipase. The results presented led to the hypothesis that in the area (336-449) a part of the active-site structure of the lipase molecule is included. It would seem that fragment (336-449) is a functional domain of lipase.