An important aspect of lipolytic enzymes is the unique physicochemical character of the reactions they catalyse at lipid-water interfaces, involving interfacial adsorption and subsequent catalysis
...sensu stricto. Lipases are now used as catalysts in aqueous as well as low-water media and accept various molecules as substrates. They were previously defined in kinetic terms, based on ‘interfacial activation’. This phenomenon was not found among esterases. Recently determined 3D structures of some, but not all, upases show a ‘lid’ controlling access to the active site. Thus, the presence of a lid, and ‘interfacial activation’, are unsuitable criteria for classifying specific esterases. Consequently, lipases can be pragmatically redefined as carboxyl-esterases acting on long-chain acylglycerols: they are simply fat-splitting ‘ferments’.
Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in ...insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.
The inhibitory effects of tetrahydrolipstatin (THL) on the hydrolytic activity of human pancreatic lipase (HPL) and
T. lanuginosa lipase (TLL) on various lipidic substrates ‘poisoned’ with THL as ...previously described was studied, using either the pH-stat, monomolecular film or oil drop technique.
Prior to adding lipase (method C), an ethanolic solution of THL was injected in a tributyrin (TC4) or a purified soybean oil (PSO) emulsion prepared in a pH-stat vessel. Under these conditions, THL was found to be a potent HPL inhibitor. After being dissolved in the pure triglyceride phase (method D), THL also strongly inhibited HPL. However, with TC4 as substrate TLL was efficiently inhibited by THL only when method C was used and not method D. The very different inhibitory effects on HPL and TLL recorded with method D and PSO as substrate were confirmed using the monomolecular film and oil drop techniques.
With a monomolecular film of dicaprin (di-C10) as substrate, 1 molecule of THL embedded in 400
000 molecules of di-C10 sufficed to reduce the HPL activity to half of its initial value.
HPL was therefore efficiently inhibited by THL with all the methods and substrates tested here. Paradoxically, TLL was inhibited by THL molecules transiently present in the aqueous phase and not by the THL molecules present at the triglyceride/water interface. It should therefore be stressed that the inhibitory effects of THL on each lipase depend strongly on the method and the substrate used.
Fat digestion in humans and some mammals such as dogs requires the successive intervention of two lipases: gastric lipase, which is stable and active despite the highly acidic stomach environment, ...followed by the classical pancreatic lipase secreted into the duodenum. We previously solved the structure of recombinant human gastric lipase (HGL) at 3.0-Å resolution in its closed form; this was the first structure to be described within the mammalian acid lipase family. Here we report on the open structure of the recombinant dog gastric lipase (r-DGL) at 2.7-Å resolution in complex with the undecyl-butyl (C11Y4) phosphonate inhibitor. HGL and r-DGL show 85.7% amino acid sequence identity, which makes it relevant to compare the forms from two different species. The open r-DGL structure confirms the previous description of the HGL catalytic triad (Ser153, His353, and Asp324) with the catalytic serine buried and an oxyanion hole (NH groups of Gln154 and Leu67). In r-DGL, the binding of the C11Y4 phosphonate inhibitor induces part of the cap domain, the lid, to roll over the enzyme surface and to expose a catalytic crevice measuring ∼20 × 20 × 7 Å3. The C11Y4 phosphonate fits into this crevice, and a molecule of β-octyl glucoside fills up the crevice. The C11Y4 phosphonate inhibitor and the detergent molecule suggest a possible binding mode for the natural substrates, the triglyceride molecules.
The inhibition of digestive lipases by the antiobesity drug Orlistat along with lipolysis levels and fecal fat excretion were measured in healthy humans. Orlistat was found to be a powerful gastric ...lipase inhibitor, achieving 46.6--91.4% enzyme inhibition and thus greatly reducing gastric lipolysis of solid and liquid meals (11--33% of respective controls). Gastric lipase inhibition by Orlistat was extremely fast (half-inhibition time < 1 min). Duodenal lipolysis was reduced significantly by Orlistat given with the solid meal (32.6--37.6% of controls) but was only slightly reduced by Orlistat given with the liquid meal (74.5--100% of controls). Human pancreatic lipase (HPL) inhibition was found to be high (51.2--82.6%), however, regardless of the meal. These paradoxical results were explained when in vitro lipolysis experiments were performed. The rates of HPL inhibition by Orlistat were found to be similar with both types of meals (half-inhibition time 5--6 min), but the preemulsified triglycerides of the liquid meal were rapidly hydrolyzed by HPL before the enzyme was significantly inhibited by Orlistat. With the solid meal, the rate of hydrolysis of the meal triglycerides by HPL was slower than the rate of HPL inhibition by Orlistat. As predicted from the previous results, the effects of Orlistat on fat excretion levels were found to be much greater with the solid (40.5--57.4% of ingested fat) than with the liquid (4.2--18.8%) test meal.
The aim of this study was to quantitatively evaluate the relative contributions to in vivo lipolysis of gastric and pancreatic lipases.
Gastric and pancreatic lipase secretions were measured, and ...their respective levels were determined in duodenal fluid during the digestion of a liquid test meal in healthy volunteers. Gastric lipase activity was clearly distinguished from that of pancreatic lipase by using both a specific enzymatic assay and an enzyme-linked immunosorbent assay. Lipolysis products were monitored throughout the digestion period.
On a weight basis, the ratio of pancreatic lipase to gastric lipase total secretory outputs was found to be around four after 3 hours of digestion. The level of gastric hydrolysis was calculated to be 10% +/- 1% of the acyl chains released from the meal triglycerides. Gastric lipase remained active in the duodenum where it might still hydrolyze 7.5% of the triglyceride acyl chains.
Globally during the whole digestion period, gastric lipase might hydrolyze 17.5% of the triglyceride acyl chains. In other words, gastric lipase might hydrolyze 1 acyl chain of 4, which need to be hydrolyzed for a complete intestinal absorption of monoglycerides and free fatty acids resulting from the degradation of two triglyceride molecules.
The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray ...crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).
Fat digestion in humans requires not only the classical pancreatic lipase but also gastric lipase, which is stable and active
despite the highly acidic stomach environment. We report here the ...structure of recombinant human gastric lipase at 3.0-Ã resolution,
the first structure to be described within the mammalian acid lipase family. This globular enzyme (379 residues) consists
of a core domain belonging to the α/β hydrolase-fold family and a âcapâ domain, which is analogous to that present in serine
carboxypeptidases. It possesses a classical catalytic triad (Ser-153, His-353, Asp-324) and an oxyanion hole (NH groups of
Gln-154 and Leu-67). Four N -glycosylation sites were identified on the electron density maps. The catalytic serine is deeply buried under a segment consisting
of 30 residues, which can be defined as a lid and belonging to the cap domain. The displacement of the lid is necessary for
the substrates to have access to Ser-153. A phosphonate inhibitor was positioned in the active site that clearly suggests
the location of the hydrophobic substrate binding site. The lysosomal acid lipase was modeled by homology, and possible explanations
for some previously reported mutations leading to the cholesterol ester storage disease are given based on the present model.
We have purified an enzyme from porcine liver microsomes which catalyzes hydrolysis of triacylglycerols. The enzyme was solubilized from the membranes by the zwitterionic detergent ...3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS) and was purified to apparent homogeneity by sequential chromatography on Q-Sepharose, hydroxyapatite, Affi-Gel heparin, and Mono-Q. The purified hydrolase migrated in SDS−polyacrylamide gel electrophoresis (PAGE) as a single polypeptide band of an apparent molecular mass of 60 kDa. The enzyme hydrolyzed long-, medium-, and short-chain triacylglycerols, as well as a chromogenic lipase substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. The highest specific activity was obtained with tributyroylglycerol (240 μmol·min-1·mg-1). The reaction rate was maximal at pH 8.5. Sulfhydryl-directed reagents, such as N-ethylmaleimide (NEM), 5,5‘-dithiobis(2-nitrobenzoic acid) (DTNB), and dodecyldithio-5-(2-nitrobenzoic acid) (C12-TNB) had no effect on the hydrolase activity; however, the enzyme was sensitive to HgCl2. Serine reagents, such as diethyl-p-nitrophenyl phosphate (E600) and diisopropyl fluorophosphate (DFP), used in 100-fold molar excess completely inhibited the activity, suggesting that it is a serine esterase. These results suggest that the enzyme may participate in the intracellular neutral lipid metabolism since the enzyme is located in the endoplasmic reticulum, an organelle where de novo triacylglycerol synthesis and assembly of lipoproteins take place.
Carboxylester hydrolases, commonly named esterases, consist of a large spectrum of enzymes defined by their ability to catalyze the hydrolysis of carboxylic ester bonds and are widely distributed ...among animals, plants, and microorganisms. Lipases are lipolytic enzymes which constitute a special class of carboxylic esterases capable of releasing long-chain fatty acids from natural water-insoluble carboxylic esters. However, up to now, several unsuccessful attempts aimed at differentiating “lipases” from “esterases” by using various criteria. These criteria were based on the first substrate used chronologically, primary sequence comparisons, some kinetic parameters, or some structural features.
Lipids are biological compounds which, by definition, are insoluble in water. Taking into account this basic physico-chemical criterion, we primarily distinguish lipolytic esterases (L, acting on lipids) from nonlipolytic esterases (NL, not acting on lipids). In view of the biochemical data accumulated up to now, we proposed a new classification of esterases based on various criteria of physico-chemical, chemical, anatomical, or cellular nature. We believe that the present attempt matters scientifically for several reasons: (1) to help newcomers in the field, performing a few key experiments to figure out if a newly isolated esterase is lipolytic or not; (2) to clarify a debate between scientists in the field; and (3) to formulate questions which are relevant to the still unsolved problem of the structure–function relationships of esterases.
PDF
