Inhibition of lipases by proteins. A kinetic study with dicaprin monolayers

We report further investigations on protein inhibition of pancreatic and microbial lipases carried out with the monolayer technique. When beta-lactoglobulin A, melittin, serum albumin, myoglobin, and a protein inhibiting lipase from soybean were preincubated with a dicaprin film at a surface pressure of 35 dynes/cm, no activity was detected with horse pancreatic or Rhizopus delemar lipases. By contrast, Rhizopus arrhizus and Geotrichum candidum lipase activities were not impaired under the same conditions. Experiments using mixed lipid-protein film transfer clearly show that the inhibition of pancreatic lipase is due to the protein associated with lipid and not caused by direct protein-enzyme interaction in the aqueous phase. Three parameters were used to determine the surface properties of the various proteins at the dicaprin/water interface; namely, the initial rate of surface pressure increase, (delta pi/delta t)t = 0, the maximal surface pressure increase, delta pi max, and the critical surface pressure, pi c. A positive correlation was observed between values of (delta pi/delta t)t = 0 of proteins and their respective capacity to inhibit pancreatic and R. delemar lipases. By contrast, there was no apparent correlation with the two other parameters, delta pi max or pi c.     

Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.     

Green tea extract (AR25) inhibits lipolysis of triglycerides in gastric and duodenal medium in vitro

In this study, we aimed to evaluate in vitro the inhibitory activity of a green tea extract (AR25 standardized at 25% catechins) on gastric and pancreatic lipase activities. We first used tributyrin as a substrate to evaluate the capability of AR25 to induce digestive lipase inhibition. Gastric lipase was totally inhibited by 40 mg AR25/g tributyrin whereas pancreatic lipase inhibition was maximum (78.8 +/- 0.7%) with 80 mg AR25/g tributyrin. We then used triolein, a long-chain triglyceride, to check whether AR25 could alter lipase activities on a physiologic substrate. AR25 60 mg/g triolein induced a dramatic inhibition of gastric lipase (96.8 +/- 0.4%) whereas pancreatic lipase activity was partially reduced (66.50 +/- 0.92%). Finally, the concerted action of gastric and pancreatic lipases was studied with an excess of enzymes to mimic the physiologic conditions observed in vivo. Incubation of AR25 with an excess of digestive lipases resulted in a drastic decrease in gastric lipolysis but the inhibitory effect on pancreatic lipase was less marked. On the whole, as compared to the control, lipolysis of triolein under the successive action of the two digestive lipases was reduced by 37 +/- 0.6% in the presence of AR25. Because a lipid/water interface is necessary for lipolysis to occur, lipid emulsification and emulsion droplet size were measured in gastric and duodenal media in the presence of AR25. In gastric and duodenal conditions, AR25 inhibited the lipid emulsification process. From these data we conclude that (1) in vitro, fat digestion is significantly inhibited by 60 mg AR25/g triolein, and (2) gastric as well as pancreatic lipase inhibition could be related to altered lipid emulsification in gastric or duodenal media. The green tea extract AR25 exhibiting marked inhibition of digestive lipases in vitro is likely to reduce fat digestion in humans.     

An Antibody to the Castor Bean Glyoxysomal Lipase (62 kD) also Binds to a 62 kD Protein in Extracts from Many Young Oilseed Plants

An antibody raised against purified glyoxysomal lipase (triacylglycerol hydrolase EC 3.1.1.3.) from castor bean (relative molecular weight of 62,000) also binds to a protein with a relative molecular weight of 62,000 in extracts of food reserve tissues from many young oilseed plants. These plants include Brassica napus L., Zea mays L., Arachis hypogaea L., Glycine max L., Gossipium hirsutum L., Cucurbita pepo L., Helianthus annuus L., Pisum sativum L., and Cicer arietinum L. The antibody caused inhibition of triacylglycerol hydrolysis by the lipases in extracts from seedlings of corn, oilseed rape, castor bean, soybean, and peanut. The pattern of antilipase binding to the 62 kilodalton protein in subcellular fractions from these other seedlings was consistent with the patterns of lipase activity reported in the literature and it is suggested that lipases from these oil seeds all have a subunit with a molecular weight of 62,000. The protein was only found in the food reserve tissues and was not present in extracts of roots and leaves of mature plants. In addition, the immunoreactive 62 kilodalton polypeptide was not detectable in lima beans and only at very low levels in kidney beans. Both these seeds are known to contain very little storage lipid and would not be expected to contain lipase. With the exception of the acid lipase of castor bean, ungerminated seeds do not generally contain active lipases. The immunoreactive 62 kilodalton protein could not be detected in the ungerminated seeds of most plants and only at very low low levels in others.     

Biochemical characterization,cloning and molecular modeling of a digestive lipase from red seabream (Pagrus major): Structural explanation of the interaction deficiency with colipase and lipidic interface

Red seabream digestive lipase (RsDL) was purified from fresh pyloric caeca. Pure RsDL has an apparent molecular mass of 50 kDa. The RsDL is more active on short‐chain triacylglycerols (TC₄), and enzymatic activity decreases when medium (TC₈) or long‐chain (olive oil) triacylglycerols were used as substrates. The specific activities of RsDL are very weak as compared to those obtained with classical pancreatic lipases. No colipase was detected in the red seabream pyloric caeca. Furthermore, the RsDL was not activated by a mammal colipase. Similar results were reported for annular seabream lipase. In order to explain structurally the discrepancies between sparidae and mammal lipases, genes encoding mature RsDL and five other lipases from sparidae fish species were cloned and sequenced. Phylogenetic studies indicated the closest homology of sparidae lipases to bird pancreatic ones. Structural models were built for annular seabream and RsDL under their closed and open forms using mammal pancreatic lipases as templates. Several differences were noticed when analyzing the amino acids corresponding to those involved in HPL binding to colipase. This is likely to prevent interaction between the fish lipase and the mammalian colipase and may explain the fact that mammalian colipase is not effective in activating sparidae lipases. In addition, several hydrophobic residues, playing a key role in anchoring pancreatic lipase onto the lipid interface, are replaced by polar residues in fish lipases. This might explain the reason why the latter enzymes display weak activity levels when compared to mammalian pancreatic lipases.     

Comparative Analyses of Lipoprotein Lipase,Hepatic Lipase,and Endothelial Lipase,and Their Binding Properties with Known Inhibitors

The triglyceride lipase gene subfamily plays a central role in lipid and lipoprotein metabolism. There are three members of this subfamily: lipoprotein lipase, hepatic lipase, and endothelial lipase. Although these lipases are implicated in the pathophysiology of hyperlipidemia and atherosclerosis, their structures have not been fully solved. In the current study, we established homology models of these three lipases, and carried out analysis of their activity sites. In addition, we investigated the kinetic characteristics for the catalytic residues using a molecular dynamics simulation strategy. To elucidate the molecular interactions and determine potential key residues involved in the binding to lipase inhibitors, we analyzed the binding pockets and binding poses of known inhibitors of the three lipases. We identified the spatial consensus catalytic triad “Ser-Asp-His”, a characteristic motif in all three lipases. Furthermore, we found that the spatial characteristics of the binding pockets of the lipase molecules play a key role in ligand recognition, binding poses, and affinities. To the best of our knowledge, this is the first report that systematically builds homology models of all the triglyceride lipase gene subfamily members. Our data provide novel insights into the molecular structures of lipases and their structure-function relationship, and thus provides groundwork for functional probe design towards lipase-based therapeutic inhibitors for the treatment of hyperlipidemia and atherosclerosis.     

Lipases from the genus Rhizopus: Characteristics,expression, protein engineering and application

Lipases are versatile catalysts that hydrolyze ester bonds of water-insoluble glycerides or carry out reversible reactions at the water/lipid interface. The remarkable characteristics of lipases from the genus Rhizopus are their high sn-1,3-positional specificity, enantioselectivity and activity in nonaqueous media, which make them one of the most desirable enzymes for many applications, including lipid modification and biodiesel and chiral organic compound synthesis. sn-1,3-Position-specific Rhizopus lipases are particularly useful for the production of structured triacylglycerols. Significant progress has been made regarding lipases from the genus Rhizopus, including gene sequencing, elucidation of the protein structure and catalytic function, heterologous expression and redesigning Rhizopus lipases for valuable properties, which is receiving increasing academic and industrial attention. In this review, we present a comprehensive overview of Rhizopus lipases, focusing on (a) the characteristics of Rhizopus lipases, (b) Rhizopus lipase genes and structural features, (c) strategies for heterologous expression of Rhizopus lipase genes in yeast system, (d) progress in protein engineering for the improvement of the properties of Rhizopus lipases, and (e) development of biotechnological applications.     

Elucidation of different inhibition mechanism of small chemicals on PtdInsP-binding domains using in silico docking experiments

Phosphatidylinositides, most negatively charged lipids in cellular membranes, regulate diverse effector proteins through the interaction with their lipid binding domains. We have previously reported inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P₃ and Btk PH domain. Here, we report that the inhibitory effects of same sets of chemicals on Grp1 PH domain and epsin1 ENTH domain to elucidate diversity of inhibitory mechanisms upon different lipid binding domains. Among the chemicals, chemical 8 showed best inhibition in vitro assay for Grp1 PH domain and epsin1 ENTH domain, and then the interaction between small chemicals and lipid binding domains was further investigated by in silico docking experiments. As a result, it was concluded that the diverse inhibitory effects on different lipid binding domains were dependent on not only the number of interactions between small chemical and domain, but also additional interaction with positively charged surfaces as the secondary binding sites. This finding will help to develop lipid binding inhibitors as antagonists for lipid–protein interactions, and these inhibitors would be novel therapeutic drug candidates via regulating effector proteins involved in severe human diseases.     

Purification and characterization of extracellular lipases from Ophiostoma piliferum

Interest in lipases from microorganisms, animals, and plants has greatly increased in the past decade due to their applications in biotransformations and organic syntheses. We are reporting the purification and characterization of two lipases from the fungus, Ophiostoma piliferum, a saprophytic organism commonly found on wood. A major and a minor lipase have been co-purified by hydrophobic interaction chromatography on octyl sepharose FF, followed by ion exchange chromatography on Q sepharose FF. The lipases bound very tightly to octyl sepharose resulting in greater than 100-fold purification in this one step. The major lipase has a molecular weight of approximately 60 kDa, a pI of 3.79, and is glycosylated as determined by PAS staining. The minor lipase, which composes 10% of the total protein, has a pI of 3.6, and molecular weight of approximately 52 kDa and did not stain with the PAS reagent. Deglycosylation of the major lipase produced two proteins of lower molecular weight, a 55 kDa protein and a 52 kDa protein. The deglycosylated protein at 52 kDa co-migrates with the minor lipase on SDS-PAGE gels. N-terminal amino acid sequencing of the major and minor lipases indicated both lipases have the same N-termini and MALDI-TOF mass spectral analysis showed similar peptide patterns. Available data indicate that the lipases are derived from the same protein and appear to differ in their post-translational modification as evidenced by their pIs and molecular weight difference. The pH rate profile and thermal stability were determined for the purified O. piliferum lipase and were consistent with a mesophilic lipase. In aqueous solution, the lipases exhibited a higher rate of hydrolysis for p-nitrophenylbutyrate (C4) than for p-nitrophenylstearate (C18), which is an unexpected result.     

Role of a sulfhydryl group in gastric lipases. A binding study using the monomolecular-film technique

Native human and rabbit gastric lipases (HGL and RGL, respectively) were inactivated after modification of one sulfhydryl group/enzyme molecule. HGL and RGL were covalently labeled using 5,5'-dithiobis(2-nitro-[14C]benzoic acid) and the interaction of 2-nitro-5-thio-[14C]benzoic-acid-labeled lipases ([14C]Nbs-lipases) with monomolecular lipid films was investigated. Our results show that [14C]Nbs-lipases bind to lipid films as efficiently as native HGL or RGL. The critical surface pressure pi c and the maximal surface pressure (delta pi max) of [14C]Nbs-lipases were enhanced in comparison with those of native RGL and HGL. These changes in behavior were probably due to an increase in hydrophobicity brought about, directly or indirectly, by the binding of the Nbs radical. This chemical modification thus blocks the hydrolysis site and reinforces the hydrophobic character of the gastric lipases.     

Specificity of the lipid-binding domain of apoC-II for the substrates and products of lipolysis

Functional similarities between colipase and apolipoprotein C-II (apoC-II) in activating lipases suggest that apoC-II may, like colipase, preferentially interact with interfaces containing the substrates and products of lipolysis. To test this hypothesis, the binding of a peptide comprising residues of the cofactor implicated in lipid binding, apolipoprotein C-II(13-56), and, to a lesser extent, apoC-II, to monomolecular lipid films was characterized. The lipids used were a diacylphosphatidylcholine, a diacylglycerol, and a fatty acid. The peptide had an affinity for the argon-buffer interface and for all lipids consistent with a dissociation constant of <10 nM. Changes in surface pressure accompanying peptide binding were comparable to those reported for native apoC-II and indicate peptide miscibility with each of the lipids tested. The capacity of the surfaces to accommodate the peptide decreased with increasing lipid concentration in the interface, indicating competition between lipid and peptide for interfacial occupancy. At a lipid acyl chain density of 470 pmol/cm2, or 35 A2 per acyl chain, a lower limit of peptide adsorption was reached with all lipids. The limiting level of adsorption to phosphatidylcholine was only 1 pmol/cm2 compared with 6;-7 pmol/cm2 for fatty acid and diacylglycerol. Similar results were obtained with apoC-II.The difference in the extent of protein adsorption to lipid classes suggests that the distribution of apoC-II among lipoproteins will depend on their lipid composition and surface pressure.     

Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer

Enantiomerically pure alkylphosphonate compounds RR′P(O)PNP (R=C_nH_2n+1, R′=OY with Y=C_n′H_2n′+1 with n=n′ or n≠n′; PNP=p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolecular films technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simultaneously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious correlation between the inhibitor molar fraction (₅₀) leading to half inhibition, and the chain length, R or Y was observed. (R₁₁Y₁₆)s were the best inhibitor of HPL and (R₁₀Y₁₁)s were the best inhibitors of HGL. We observed a highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a scalemic mixture. We also showed, for the first time, that this enantioselective recognition can occur either during the catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric inhibitors.     

Inhibition of human pancreatic lipase by tetrahydrolipstatin: Further kinetic studies showing its reversibility

Tetrahydrolipstatin (THL, Orlistat) is a potent inhibitor of gastrointestinal lipases. Using the pH-stat technique we report that, in the absence of substrate, THL (at a molar excess of 100) inhibits rapidly (after few minutes of incubation) human pancreatic lipase (HPL). Bile salts over their critical micellar concentration (CMC) were found to accelerate the inhibition process.At variance with the generally accepted model of a covalent and quasi-irreversible acyl-lipase complex, we showed here that the inhibition of HPL could be rapidly and partially reversed in the presence of an emulsion of short- or long-chain triacylglycerols, as indicated by a kinetic reactivation process. The presence of bile salts in the incubation medium, containing THL and HPL, was found to stabilise the covalent complex as reflected by a decrease in the reactivation rate. Paradoxically, the presence of bile salts in the lipase assay enhanced this reactivation process probably by forming mixed micelles between bile salts and THL, which accelerates the deacylation phenomenon.On the basis of this kinetic study, a general model is proposed to describe the inhibition of lipases by THL in the aqueous phase as well as its partial reactivation process at the lipid–water interface.     

Lipid-lipid interactions as regulators of carboxylester lipase activity

The hydrolysis of 1,3-dioleoylglycerol and related substrates by mammalian pancreatic carboxylester lipases was studied. Mixed lipid films of substrates with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine at the argon-buffer interface were exposed to relatively high levels of monomeric porcine pancreatic carboxylester lipase for a brief period. With either 1,3-dioleoylglycerol, 1,2-dioleoylglycerol, trioleoylglycerol, or oleoylmethanol as a substrate, the percentage of substrate hydrolysis increased abruptly from near zero to near 100% with increasing proportion of substrate in the film. The phospholipid was not hydrolyzed. Using 1,3-dioleoylglycerol as the substrate with either the dimeric, porcine pancreatic carboxylester lipase, human pancreatic carboxylester lipase, or human milk bile salt-stimulated lipase gave results identical to those obtained with the porcine monomer. Hydrolysis of 1,3-dioleoylglycerol by porcine monomeric carboxylester lipase was independent of the initial surface pressure of the film. However, a strong correlation was observed between hydrolysis and interfacial lipid composition at all surface pressures, even if bulk 1,3-dioleoylglycerol was also present. The ultrasensitive dependence of hydrolysis on interfacial lipid composition, i.e. lipid-lipid interactions, suggests that such "switching" may contribute to the regulation of diacylglycerol levels in cells where they function in signal transduction.     

Inhibitors of lipase activities in soybean and other oil seeds

In the cotyledon extracts of seedlings of many oil seeds, including soybean, sunflower, cucumber, and peanut, the in vitro lipase activity was too low to account for the observed in vivo lipolysis. The low in vitro lipase activity was due to the presence of lipase inhibitors in the extracts. The inhibitors from soybean were characterized based on their effects on the hydrolysis of trilinolein by corn, pancreatic, and Rhizopus lipases. The inhibitors were not dialyzable and unaltered by RNase and β-galactosidase treatment. However, they were sensitive to heating and protease digestion. The inhibitory effect of the inhibitors was expressed irrespective of the sequence of the addition of lipase, substrate, and inhibitors to the assay medium. The inhibitory effect was equally expressed when the inhibitors were added either before or after the lipase reaction had been in progress. The inhibitory effect of the inhibitors was independent of the amount of lipase present in the assay, but was dependent on the amount of substrate added. High substrate concentration eliminated totally the inhibitory effect of the inhibitors. Most of the inhibitors were recovered in the soluble fraction in subcellular fractionation. They were present in the 2-4S and not in the 7S, and 11S (storage proteins) protein fraction. There was a gradual decrease of the inhibitors in the cotyledons in the postgerminative growth. We suggest that the inhibitors are proteins which bind to the surface of the substrate micelles. The binding prevents the normal functioning of lipase which acts on the interfacial area between the aqueous medium and the micelle surface.     

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.     

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis.

In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.     

Interaction of cytochrome c with lipid monolayers

Cytochrome c was permitted to react with several lipid monolayers in which surface pressure, lipid charge and unsaturation were varied. Cytochrome c interaction with the films caused increased surface pressures, and the magnitude and rate of surface pressure change were compared under a variety of experimental conditions. Large surface pressure changes were associated with more expanded films, whereas greater rates of surface pressure change were associated with favorable charge interaction between cytochrome c and the films. Under the most favorable conditions, rates of surface pressure change were limited principally by protein diffusion to the interface. From these data, it is suggested that unsaturation in lipids of biological membranes may help stabilise non-polar protein-lipid interactions, whereas charge interaction may facilitate and direct initial binding of protein to membranes.     

Exploring the conformational states and rearrangements of Yarrowia lipolytica Lipase

We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.     

Solvent-induced lid opening in lipases: A molecular dynamics study

In most lipases, a mobile lid covers the substrate binding site. In this closed structure, the lipase is assumed to be inactive. Upon activation of the lipase by contact with a hydrophobic solvent or at a hydrophobic interface, the lid opens. In its open structure, the substrate binding site is accessible and the lipase is active. The molecular mechanism of this interfacial activation was studied for three lipases (from Candida rugosa, Rhizomucor miehei, and Thermomyces lanuginosa) by multiple molecular dynamics simulations for 25 ns without applying restraints or external forces. As initial structures of the simulations, the closed and open structures of the lipases were used. Both the closed and the open structure were simulated in water and in an organic solvent, toluene. In simulations of the closed lipases in water, no conformational transition was observed. However, in three independent simulations of the closed lipases in toluene the lid gradually opened. Thus, pathways of the conformational transitions were investigated and possible kinetic bottlenecks were suggested. The open structures in toluene were stable, but in water the lid of all three lipases moved towards the closed structure and partially unfolded. Thus, in all three lipases opening and closing was driven by the solvent and independent of a bound substrate molecule.