orlistat and tributyrin

Tetrahydrolipstatin is known as an inhibitor for pancreatic lipase but not for microbial lipases. In this paper we demonstrate that in the presence of water-insoluble substrates like tributyrin or olive oil, tetrahydrolipstatin inhibits the lipases of Chromobacterium viscosum and Rhizopus oryzae, although with different potency. In contrast to porcine pancreatic lipase, which forms an irreversible and covalent enzyme-inhibitor complex with tetrahydrolipstatin, the inhibition of the microbial lipases is reversible as the inhibitor can be removed from the enzyme-inhibitor complex by solvent extraction. Moreover, after inhibition of Chromobacterium viscosum lipase tetrahydrolipstatin remains chemically unchanged.

Topics: 2-Propanol; Chromatography, Thin Layer; Chromobacterium; Emulsions; Enzyme Activation; Enzyme Inhibitors; Kinetics; Lactones; Lipase; Molecular Structure; Olive Oil; Orlistat; Plant Oils; Protein Binding; Rhizopus; Triglycerides

Lipoprotein lipase (LPL) was rapidly inactivated by low concentrations of the active-site inhibitor tetrahydrolipstatin (THL). The presence of amphiphils (e.g. long-chain fatty acids) or of lipid/water interfaces (lipid emulsions) was required for inhibition to occur. Apolipoprotein CII increased the maximal inactivation rate constant by 1.8-fold in the presence of an emulsion of long-chain triacylglycerols, but had no effect in the presence of an emulsion of tributyrylglycerol. The fully inhibited enzyme had a ratio of THL/LPL of nearly 2, indicating that both subunits of the LPL homo-dimer bound THL. The THL-LPL complex was stable below pH 7.5. At higher pH reactivation occurred indicating that THL was slowly turned over by the enzyme. The apparent reactivation rate constant was increased about threefold by the presence of lipid/water interfaces. Sucrose density gradient centrifugation revealed that THL induces tetramerisation of LPL. This aggregation was reversible on reactivation of the inhibited enzyme. Binding to heparin was not affected by THL. In contrast, binding to lipid droplets and to lipoproteins was increased, indicating exposure of hydrophobic regions in the inhibited LPL. It is suggested that THL induces local conformational changes in LPL, which may involve opening of the putative surface lid structure which covers the active-site.

Topics: Animals; Apolipoprotein C-II; Apolipoproteins C; Binding Sites; Chromatography, Affinity; Chylomicrons; Deoxycholic Acid; Enzyme Activation; Fat Emulsions, Intravenous; Kinetics; Lactones; Lipoprotein Lipase; Lymph; Macromolecular Substances; Orlistat; Protein Binding; Rats; Triacetin; Triglycerides; Triolein

Tetrahydrolipstatin is a specific lipase inhibitor derived from lipstatin, a lipid produced by Streptomyces toxytricini. In addition to pancreatic lipase, it is shown in the present study that tetrahydrolipstatin also inhibits human gastric lipase, carboxyl ester lipase (cholesterol esterase) of pancreatic origin and the closely related bile-salt-stimulated lipase of human milk. It does not inhibit the exocellular lipase from Rhizopus arrhizus or a lipase recently isolated from Staphylococcus aureus. In the presence of a water-insoluble substrate, such as tributyrin, the inhibition has the characteristics of an irreversible inactivation of the uncompetitive type, thus indicating that an enzyme.substrate.inhibitor complex is formed, which cannot undergo further reaction to yield the normal product. This reaction probably takes place at the aqueous/oil interface of the substrate. In aqueous solution, in the absence of substrate, the inhibition of carboxyl ester lipase by tetrahydrolipstatin has the characteristics of being reversible, and finally becomes of a temporary nature analogues to the trypsin-trypsin inhibitor system. It is suggested that an enzyme-inhibitor complex of an acyl-enzyme type is formed that is slowly hydrolysed, with water as the final acceptor, leaving an intact enzyme and an inactive form of the inhibitor. The enzyme thus consumes the inhibitor, which undergoes a chemical conversion, as indicated by a change in mobility in an appropriate thin-layer chromatographic system, indicating an increase in hydrophilicity. Evidence is presented that the reaction product is an acid and that the functional group of tetrahydrolipstatin is the beta-lactone reacting with the active site of the enzyme.

Topics: Bile Acids and Salts; Binding Sites; Kinetics; Lactones; Lipase; Milk, Human; Orlistat; Pancreas; Pancreatic Juice; Rhizopus; Solutions; Staphylococcus aureus; Sterol Esterase; Stomach; Triglycerides