Competitive inhibitory effect exerted by bile salt micelles on the hydrolysis of tributyrin by pancreatic lipase.

Colipase was readily adsorbed on tributyrin particles emulsified with sodium taurodeoxycholate used above its critical micellar concentration. The ensuing rate of lipase adsorption on tributyrin depended on the amount of colipase already adsorbed at the interface. A molar excess of colipase over lipase was required to observe lipolysis at maximal velocity. Bile salt micelles competitively inhibited this reaction.     

Effects of gum arabic on lipase interfacial binding and activity.

We investigated the surface behavior of gum Arabic (GA) as well as its effects on the lipolytic activity of human pancreatic lipase (HPL) and Humicola lanuginosa lipase (HLL), using emulsions of triacylglycerols (TAG) with various chain lengths. The effects of GA on the interfacial binding of HPL were also investigated. In the presence of 4 mM sodium taurodeoxycholate (NaTDC), GA (3% w/v, final concentration) had no effect on the HPL activity measured in the presence of colipase, whatever the type of TAG used. However, in the absence of bile salts or at low bile salt concentrations, GA inhibited the HPL activity when trioctanoin (TC8) and purified soybean oil (PSO) were used as substrates. At 3% (w/v, final concentration), GA strongly desorbed pure HPL from the TC8 interface and the classical anchoring effect of colipase was clearly observed. Both crude and dialyzed GA solutions were found to be highly tensioactive at the air-water as well as the oil-water interface using the drop technique. In conclusion, GA, or a putative contaminant present in GA, was found to be surface active and to have similar effects to those of bile salts on the interfacial binding and activity of HPL.     

Inhibition of lipase adsorption at interfaces. Role of bile salt micelles and colipase

The effects of bile salts and colipase on the adsorption of lipase at an interface were studied by hydrophobic affinity chromatography on phenyl- and octyl-Sepharose. In the absence of bile salts, lipase or colipase binds separately to the gel. This is unchanged in the presence of adsorbed bile salts, when one bile salt molecule is associated per hydrophobic ligand. The same data are obtained in the presence of monomeric bile salt solutions. In contrast, lipase adsorption is totally prevented in a micellar bile salt solution. These results favor the idea that the formation of a lipase-bile salt complex in solution is responsible for the lack of interfacial lipase adsorption.     

On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts.

The interactions between pancreatic lipase and colipase and the substrate and the effect of bile salts on these interactions have been investigated by the use of kinetic experiments and studies on the semiquantitative phase distribution of lipase and colipase activities. The results suggest that lipase binds to hydrophobic interfaces with partial irreversible inactivation. Bile salts in the range of micellar concentrations and above a pH of about 6.5 displace lipase from this binding, resulting in a reversible in activation. At pH values below about 6.5, lipase binds strongly to the substrate even in the presence of bile salt, and a low activity peak is seen around pH 5.5. This is the result of the binding of lipase to the "supersubstrate" and the activity of the catalytic site. In the presence of bile salt, colipase promotes the binding of lipase to the "supersubstrate" but not to other hydrophobic interfaces, and catalytic activity is reestablished. Kinetic data indicate that the binding between colipase and lipase in the presence of substrate is strong and occurs in an approximately stoichiometric relationship.     

Human pancreatic lipase: colipase dependence and interfacial binding of lid domain mutants

Five key amino acid residues from human pancreatic lipase (HPL) are mutated in some pancreatic lipase-related proteins 2 (PLRP2) that are not reactivated by colipase in the presence of bile salts. One of these residues (Y403) is involved in a direct interaction between the HPL C-terminal domain and colipase. The other four residues (R256, D257, Y267, and K268) are involved in the interactions stabilizing the open conformation of the lid domain, which also interacts with colipase. Here we produced and characterized three HPL mutants: HPL Y403N, an HPL four-site mutant (R256G, D257G, Y267F, and K268E), and an HPL five-site mutant (R256G, D257G, Y267F, K268E, and Y403N), in which the HPL amino acids were replaced by those present in human PLRP2. Colipase reactivated both the HPL Y403N mutant and HPL, and Y403 is therefore not essential for lipase-colipase interactions. Both the HPL four-site and five-site mutants showed low activity on trioctanoin, were inhibited by bile salts (sodium taurodeoxycholate, NaTDC) and were not reactivated by colipase. The interfacial binding of the HPL four-site mutant to a trioctanoin emulsion was suppressed in the presence of 4 mM NaTDC and was not restored by addition of colipase. Protein blotting/protein overlay immunoassay revealed that the HPL four-site mutant-colipase interactions are not abolished, and therefore, the absence of reactivation of the HPL four-site mutant is probably due to a lid domain conformation that prevents the interfacial binding of the lipase-colipase complex. The effects of colipase were also studied with HPL(-lid), an HPL mutant showing an 18-residue deletion within the lid domain, which therefore has only one colipase interaction site. HPL(-lid) showed a low activity on trioctanoin, was inhibited by bile salts, and recovered its lipase activity in the presence of colipase. Reactivation of HPL(-lid) by colipase was associated with a strong interfacial binding of the mutant to a trioctanoin emulsion. The lid domain is therefore not essential for either the interfacial binding of HPL or the lipase-colipase interactions.     

Computational study of colipase interaction with lipid droplets and bile salt micelles

Colipase is a key element in the lipase-catalyzed hydrolysis of dietary lipids. Although devoid of enzymatic activity, colipase promotes the pancreatic lipase activity in physiological intestinal conditions by anchoring the enzyme at the surface of lipid droplets. Analysis of structures of NMR colipase models and simulations of their interactions with various lipid aggregates, lipid droplet, and bile salt micelle, were carried out to determine and to map the lipid binding sites on colipase. We show that the micelle and the oil droplet bind to the same side of colipase 3D structure, mainly the hydrophobic fingers. Moreover, it appears that, although colipase has a single direction of interaction with a lipid interface, it does not bind in a specific way but rather oscillates between different positions. Indeed, different NMR models of colipase insert different fragments of sequence in the interface, either simultaneously or independently. This supports the idea that colipase finger plasticity may be crucial to adapt the lipase activity to different lipid aggregates.     

The effect of pancreatic procolipase and colipase on pancreatic lipase activation

Intestinal fat digestion is carried out by the concerted action of pancreatic lipase and its protein cofactor colipase. Colipase is secreted from pancreas as a procolipase and is transformed into colipase by the trypsin cleavage of the Arg5-Gly6 bond during liberation of an N-terminal pentapeptide. The kinetic parameters for the lipase-colipase system compared to the lipase-procolipase system has been compared using trioctanoin and Intralipid as substrates. It was found that at pH 7.0 the Kmapp using Intralipid as substrate was the same for procolipase and colipase, 0.06 mM and 0.05 mM, respectively. At pH 8.0, however, the Kmapp were different-0.23 mM for procolipase and 0.08 mM for colipase. In a similar way the binding between colipase and lipase had a dissociation constant of 2.4 x 10(-6) M at pH 7.0, while for procolipase--lipase binding the dissociation constant was 4.1 x 10(-6) M with no significant difference. At pH 8.0 the binding between colipase and lipase was stronger, Kd being 2.0 x 10(-7) M, while weaker for procolipase and lipase, Kd being 1.0 x 10(-5) M. It is concluded that at the physiological pH value as is found in the intestine, the activation of procolipase to colipase has no influence on the hydrolysis of trioctanoin or Intralipid in the presence of bile salt.     

Rapid exchange of pancreatic lipase between triacylglycerol droplets

Two types of experiments were performed to study the reversibility of interfacial adsorption of pancreatic lipase (PL) to fat droplets during lipolysis. Lipolysis was measured in olive oil/gum arabic emulsions containing radiolabeled triolein in the presence of bile salts and lecithin at rate-limiting concentrations of porcine PL (PPL) or human PL (HPL). The lipolysis rate in a labeled emulsion, i.e. release of [(14)C]oleic acid, was immediately reduced by around 50% upon dilution with an equal amount of an unlabeled emulsion. Further, lipolysis was rapidly and completely suppressed when a non-exchanging lipase inhibitor was present in the second emulsion. These results indicate hopping of lipase between emulsion droplets. Alternative explanations were excluded. Hopping of PL between triolein droplets stabilized with gum arabic at supramicellar bile salt concentrations was observed only in the presence, not in the absence, of lecithin. Displacement from a trioctanoin-water interface of active HPL by an inactive mutant (S152G) was studied in the presence of bile salts by measuring HPL distribution between the water phase and the oil-water interface. Colipase was limiting for HPL binding to the oil-water interface (colipase to lipase molar ratio: 0.5) and, thus, for lipolysis. Upon adding S152G, which has the same affinity for colipase, inactive and active HPL were found to compete for binding at the oil-water interface. When equal amounts of HPL and HPL S152G were used, the lipolysis rate dropped to half the maximum rate recorded with HPL alone, suggesting that half the active HPL was rapidly desorbed from the oil-water interface. Therefore, under various conditions, PL does not remain irreversibly adsorbed to the oil-water interface, but can exchange rapidly between oil droplets, via an equilibrium between soluble and lipid-bound PL.     

Colipase residues Glu64 and Arg65 are essential for normal lipase-mediated fat digestion in the presence of bile salt micelles

Pancreatic triglyceride lipase (PTL) requires colipase for activity. Various constituents in meals and in bile, particularly bile acids, inhibit PTL. Colipase restores activity to lipase in the presence of inhibitory substances like bile acids. Presumably, colipase functions by anchoring and orienting PTL at the oil-water interface. The x-ray structure of the colipase.PTL complex supports this model. In the x-ray structure, colipase has a hydrophobic surface positioned to bind substrate and a hydrophilic surface, lying opposite the hydrophobic surface, with two putative lipase-binding domains, Glu(45)/Asp(89) and Glu(64)/Arg(65). To determine whether the hydrophilic surface interacts with PTL in solution, we introduced mutations into the putative PTL binding domains of human colipase. Each mutant was expressed, purified, and assessed for activity against various substrates. Most of the mutants showed impaired ability to reactivate PTL, with mutations in the Glu(64)/Arg(65) binding site causing the greatest effect. Analysis indicated that the mutations decreased the affinity of the colipase mutants for PTL and prevented the formation of PTL.colipase complexes. The impaired function of the mutants was most apparent when assayed in micellar bile salt solutions. Most mutants stimulated PTL activity normally in monomeric bile salt solutions. We also tested the mutants for their ability to bind substrate and anchor lipase to tributyrin. Even though the ability of the mutants to anchor PTL to an interface decreased in proportion to their activity, each mutant colipase bound to tributyrin to the same extent as wild type colipase. These results demonstrate that the hydrophilic surface of colipase interacts with PTL in solution to form active colipase.PTL complexes, that bile salt micelles influence that binding, and that the proper interaction of colipase with PTL requires the Glu(64)/Arg(65) binding site.     

Lipase-colipase interactions during gel filtration. High and low affinity binding situations

The interaction of porcine pancreatic lipase and colipase was studied during gel filtration in columns eluted with a variety of buffers. High and low affinity binding situations were observed under different conditions. Low affinity binding could only be detected at the high lipase-colipase concentrations encountered during batch purification (10(-3)-10(-4) M). Even in this situation the rapid dissociation of the weak complex during filtration resulted in considerable separation of the two proteins. High affinity binding of lipase to colipase was observed at protein eluant concentrations as low as 10(-8) M on columns equilibrated with oleic acid-taurodeoxycholate mixed micelles. This binding did not take place on columns equilibrated with simple bile salt and mixed phosphatidylcholine-cholesterol-bile salt micelles. Colipase alone exhibited strong binding to phosphatidylcholine and fatty acid mixed bile salt micelles when applied together in a sample on columns eluted with pure bile salt micelles, lipase did not. The relevance of the high affinity complex to the lipase . colipase . substrate complex is discussed.     

Kinetic behavior of the pancreatic lipase-colipase-lipid system

Pancreatic lipase is a surface-active protein that binds avidly to interfaces comprised of the substrates and products of lipolysis. However, both lipase binding to substrate-containing particles and subsequent interfacial catalysis are inhibited by a number of amphipathic molecules. The most thoroughly studied of these, phosphatidylcholine, is a common constituent of membranes and intestinal lipid contents. Colipase, a surface-active cofactor of lipase, relieves inhibition by phosphatidylcholine in several ways. Through protein-protein interactions, colipase helps anchor lipase to surfaces and stabilizes it in the open conformation. Within the interface, colipase packs more efficiently with substrates and products of lipolysis than with phosphatidylcholine, thereby concentrating these reactants in the vicinity of colipase. This enrichment of lipase substrates and products in the vicinity of colipase enhances lipase-lipid interactions. The result is that colipase facilitates the adsorption of lipase to the interface and, possibly, increases the availability of substrate to the enzyme. Thus, the functional unit in intestinal lipolysis appears to be a lipase-colipase-reactant complex.     

Inhibition of pancreatic and microbial lipases by proteins

We have compared the effect of several proteins, including melittin, beta-lactoglobulin A, serum albumin, ovalbumin and myoglobin, on the hydrolysis of tributyrin and triolein by lipases from various origins. All proteins tested inactivate pancreatic lipase in absence of colipase and bile salt. Inhibition is not significantly reversed by colipase in absence of bile salt except in systems containing tributyrin and melittin or triolein and beta-lactoglobulin A. In all other cases, activation of pancreatic lipase by colipase in presence of inhibitory protein requires the presence of bile salt. Lipase from Rhizopus delemar is also inhibited by the proteins that inactivate pancreatic lipase. In contrast, the activity of lipase from Rhizopus arrhizus is not affected by the proteins in the same concentration range. Inhibition of lipase activity by amphiphiles such as proteins or detergents appears to be a general phenomenon not directly related to a decrease in tension at the triacylglycerol-water interface. Inhibition could be the result of desorption of lipase from its substrate due to a change in interfacial quality.     

Inhibition of pancreatic lipase B activity by taurodeoxycholate and its reversal by colipase.

In our two-phase reaction system taurodexycholate prevents the adsorption of pancreatic lipase B to the nonaqueous phase. Our data are consistent with a mechanism for this reaction which involves the cooperative formation of an enzyme-(bile salt)4 complex in solution with a dissociation constant of 1.4 X 10(-15)M4. Whereas the free enzyme is readily adsorbed to a bile salt-substrate-covered surface, the complex is not. Thus, the "inhibition" of substrate hydrolysis occurs because enzyme and substrate are separated physically. The protein cofactor, colipase, reverses the inhibitory effects of bile salt by providing a high affinity binding site at the interface for the lipase-(bile salt)4 complex. Steady state and presteady state kinetic data are consistent with the formation of a complex with a 1/1, lipase/colipase, ratio, and a dissociation constant of 0.4 to 2.8 X 10(-9)M. The rate of adsorption of lipase to adsorbed colipase appears to be controlled by diffusion through the unstirred layer with a second order rate constant of 1.3 X 10(6)M-1S-1.     

Hydrolysis of emulsified mixtures of triacylglycerols by pancreatic lipase

Hydrolysis of the emulsified mixture of short-chain triacylglycerols by porcine pancreatic lipase in the presence of procolipase and micellar sodium taurodeoxycholate has been studied. Increase in the content of tributyrin and trioctanoin in the mixture with triacetin had highly cooperative effects on the formation of the interfacial lipase procolipase complex. Abrupt enhancement of the complex stability was observed in the presence of 0.4-0.6 mol mol-1 of tributyrin or 0.58 mol mol-1 of trioctanoin in the substrate phase. The affinity of lipase towards interfacially bound procolipase for the trioctanoin containing 0.07-0.42 mol mol-1 of triacetin was approximately three times higher than that for pure trioctanoin. The cooperative processes involved in complex formation did not contribute to the affinity of the interfacial lipase/(pro)colipase complex towards substrate molecules and its catalytic activity.     

Lipid lateral organization in fluid interfaces controls the rate of colipase association.

Colipase, a cofactor of pancreatic triacylglycerol lipase, binds to surfaces of lipolysis reactants, like fatty acid and diacylglycerol, but not to the nonsubstrate phosphatidylcholine. The initial rate of colipase binding to fluid, single-phase lipid monolayers was used to characterize the interfacial requirements for its adsorption. Colipase adsorption rates to phosphatidylcholine/reactant mixed monolayers depended strongly on lipid composition and packing. Paradoxically, reactants lowered colipase adsorption rates only if phosphatidylcholine was present. This suggests that interactions between phosphatidylcholine and reactants create dynamic complexes that impede colipase adsorption. Complex formation was independently verified by physical measurements. Colipase binding rate depends nonlinearly on the two-dimensional concentration of phosphatidylcholine. This suggests that binding is initiated by a cluster of nonexcluded surface sites smaller than the area occupied by a bound colipase. Binding rates are mathematically consistent with this mechanism. Moreover, for each phosphatidylcholine-reactant pair, the complex area obtained from the analysis of binding rates agrees well with the independently measured collapse area of the complex. The dynamic complexes between phosphatidylcholine and lipids, like diacylglycerols, exist independently of the presence of colipase. Thus, our results suggest that lipid complexes may regulate the fluxes of other proteins to membranes during, for example, lipid-mediated signaling events in cells.     

A comparative study on two fungal lipases from Thermomyces lanuginosus and Yarrowia lipolytica shows the combined effects of detergents and pH on lipase adsorption and activity

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air-water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil-water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.     

Probing the opening of the pancreatic lipase lid using site-directed spin labeling and EPR spectroscopy

Access to the active site of human pancreatic lipase (HPL) is controlled by a surface loop (the lid) that undergoes a conformational change in the presence of amphiphiles and lipid substrate. The question of how and when the lid opens still remains to be elucidated, however. A paramagnetic probe was covalently bound to the lid via the D249C mutation, and electron paramagnetic resonance (EPR) spectroscopy was used to monitor the conformational change in solution. Two EPR spectral components, corresponding to distinct mobilities of the probe, were attributed to the closed and open conformations of the HPL lid, based on experiments performed with the E600 inhibitor. The open conformation of the lid was observed in solution at supramicellar bile salt concentrations. Colipase alone did not induce lid opening but increased the relative proportions of the open conformation in the presence of bile salts. The opening of the lid was found to be a reversible process. Using various colipase to lipase molar ratios, a correlation between the proportion of the open conformation and the catalytic activity of HPL was observed.     

Structure and orientation of pancreatic colipase in a lipid environment: PM-IRRAS and Brewster angle microscopy studies

Colipase is a key element in lipase-catalyzed dietary lipids hydrolysis. Although devoid of enzymatic activity, colipase promotes pancreatic lipase activity in the physiological intestinal conditions by anchoring the enzyme on the surface of lipid droplets. Polarization modulation infrared reflection absorption spectroscopy combined with Brewster angle microscopy studies was performed on colipase alone and in various lipid environments to obtain a global view of both conformation and orientation and to assess lipid perturbations. We clearly show that colipase fully inserts into a dilaurin monolayer and promotes the formation of lipid/protein domains, whereas in a phospholipid environment its insertion is only partial, limited to the polar head group. In a mixed 70% phosphatidylcholine/30% dilaurin environment, colipase adsorbs to but does not penetrate deeply into the film. It triggers the formation of diglyceride domains under which it would form a rather uniform layer. We also clearly demonstrate that colipase adopts a preferred orientation when dilaurin is present at the interface. In contrast, at a neutral phospholipid interface, the infrared spectra suggest an isotropic orientation of colipase which could explain its incapacity to reverse the inhibitory effects of these lipids on the lipase activity.     

Role of the lid hydrophobicity pattern in pancreatic lipase activity

Pancreatic lipase is a soluble globular protein that must undergo structural modifications before it can hydrolyze oil droplets coated with bile salts. The binding of colipase and movement of the lipase lid open access to the active site. Mechanisms triggering lid mobility are unclear. The *KNILSQIVDIDGI* fragment of the lid of the human pancreatic lipase is predicted by molecular modeling to be a tilted peptide. Tilted peptides are hydrophobicity motifs involved in membrane fusion and more globally in perturbations of hydrophobic/hydrophilic interfaces. Analysis of this lid fragment predicts no clear consensus of secondary structure that suggests that its structure is not strongly sequence determined and could vary with environment. Point mutations were designed to modify the hydrophobicity profile of the [240-252] fragment and their consequences on the lipase-mediated catalysis were tested. Two mutants, in which the tilted peptide motif was lost, also have poor activity on bile salt-coated oil droplets and cannot be reactivated by colipase. Conversely, one mutant in which a different tilted peptide is created retains colipase dependence. These results suggest that the tilted hydrophobicity pattern of the [240-252] fragment is neither important for colipase binding to lipase, nor for interfacial binding but is important to trigger the maximal catalytic efficiency of lipase in the presence of bile salt.     

Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives

The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.