Hydrolysis of p-nitrophenyl acetate by the peptide chain fragment (336-449) of porcine pancreatic lipase.
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.
D000107:Acetylation; D000818:Animals; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000032:analysis*; D009596:Nitrophenols / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000494:pharmacology*; D013552:Swine
D009596:Nitrophenols; D010446:Peptide Fragments; C008642:4-nitrophenyl acetate; D008049:Lipase; D004047:Diethyl Pyrocarbonate