The kinetics of formation of inclusion complexes between beta-cyclodextrin and monolayers of one-, two- and three-chained lipid molecules, namely, oleic acid (OA), monoolein (MO), diolein (DO) and triolein (TO), was investigated at various pH using three independent dynamic methods. The formation and solubilization of soluble inclusion beta-CD/OA and beta-CD/MO complexes was detected by measuring the decrease of the surface area and surface pressure of the OA and MO monolayers in the presence of beta-CD within a wide range of concentrations. A third approach, describing the dilatational properties of the monolayers, influenced by the formation and solubilization of the complexes, was developed. Using the three above-mentioned independent methods, the rate constants of formation (k1) and dissociation (k2) of beta-CD/OA and beta-CD/MO, were determined. We observed that solubilization flux i s for OA monolayer increases with pH and at pH 11 reached a value, which is closed to the diffusion flux iD and the process thus becomes diffusion controlled. For MO monolayer no significant effects of pH was observed above pH 6. The surface pressure (Deltapi)--area per molecule (A) and surface potential (DeltaV)--area per molecule (A) isotherms and rheological properties of DO and TO monolayers were measured in the presence or absence of beta-CD. DO and TO form water-insoluble complexes with beta-CD, as visualized by AFM images.

The binding of Thermomyces lanuginosa lipase and its mutants [TLL(S146A), TLL(W89L), TLL(W117F, W221H, W260H)] to the mixed micelles of cis-parinaric acid/sodium taurodeoxycholate at pH 5.0 led to the quenching of the intrinsic tryptophan fluorescence emission (300-380 nm) and to a simultaneous increase in the cis-parinaric acid fluorescence emission (380-500 nm). These findings were used to characterize the Thermomyces lanuginosa lipase/cis-parinaric acid interactions occurring in the presence of sodium taurodeoxycholate. The fluorescence resonance energy transfer and Stern-Volmer quenching constant values obtained were correlated with the accessibility of the tryptophan residues to the cis-parinaric acid and with the lid opening ability of Thermomyces lanuginosa lipase (and its mutants). TLL(S146A) was found to have the highest fluorescence resonance energy transfer. In addition, a TLL(S146A)/oleic acid complex was crystallised and its three-dimensional structure was solved. Surprisingly, two possible binding modes (sn-1 and antisn1) were found to exist between oleic acid and the catalytic cleft of the open conformation of TLL(S146A). Both binding modes involved an interaction with tryptophan 89 of the lipase lid, in agreement with fluorescence resonance energy transfer experiments. As a consequence, we concluded that TLL(S146A) mutant is not an appropriate substitute for the wild-type Thermomyces lanuginosa lipase for mimicking the interaction between the wild-type enzyme and lipids.

The hydrolysis of 1,2-diolein (DO) monomolecular films by Humicola lanuginosa lipase (HLL) was studied by simultaneous measuring the decrease in the film area and the changes in the surface potential in the “zero-order trough” at constant surface pressure and in the presence of β-cyclodextrin (β-CD). The decrease with time in the film area reflects both the reduction in the area per molecule due to the transformation of substrate DO molecules into the products molecules of monoolein (MO) and oleic acid (OA) and the desorption of the soluble inclusion complexes β-CD–MO and β-CD–OA. The surface potential data were interpreted as an accumulation at the interface of negatively charged products of OA and insoluble β-CD–DO complexes. In the proposed kinetic model, the product solubilization rates in the presence of β-CD and the flux supplied progressively by the moving barrier from the reservoir to the reaction compartment in order to keep the constant surface pressure were taken into account. The surface concentrations of MO and OA transiently present at the interface were determined. The values of the global kinetic constant Qm′ of hydrolysis of DO to MO were obtained. Comparison with the values of the global kinetic constant of hydrolysis of monoglyceride MO to OA shows that the rates of hydrolysis of diglyceride and monoglyceride by HLL are of the same order of magnitude.

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and α-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and α-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of β-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.

The fluorescence resonance energy transfer (FRET) technique was adapted to study the process whereby lipase is adsorbed to monomolecular lipid films spread at the air-water interface. When cis-parinaric acid (cis-PnA) was spread over an aqueous subphase before the injection of sodium taurodeoxycholate (NaTDC) and Thermomyces lanuginosa lipase (TLL), no FRET was observed. Under these conditions, no adsorption of TLL was detected using an ELISA. In contrast, FRET occurred when cis-PnA was spread over an aqueous subphase containing NaTDC and TLL. The FRET signals observed were attributed to the interactions between the adsorbed TLL and the cis-PnA monomolecular films. Comparisons between the fluorescence emission spectra corresponding to the bulk phase and the aspirated film, in the presence and absence of TLL, showed that cis-PnA was undetectable in the bulk phase. We concluded that the FRET originated from the interface and not from the bulk phase. Using surface FRET, we estimated that the surface excess of the catalytically inactive mutant, TLL(S146A), was 1.6 higher than that present in the wild-type TLL. This finding is in agreement with independent measurements of the surface excess of TLL and TLL(S146A) on monomolecular films of cis-PnA.

The structural similarities between the C-terminal domain of human pancreatic lipase (C-HPL) and C2 domains suggested a similar function, the interaction with lipids. The catalytic N-terminal domain (N-HPL) and C-HPL were produced as individual proteins, and their partitioning between the water phase and the triglyceride-water interface was assessed using trioctanoin emulsions (TC8). N-HPL did not bind efficiently to TC8 and was inactive. C-HPL did bind to TC8 and to a phospholipid monolayer with a critical surface pressure of penetration similar to that of HPL (15 mN m(-1)). These experiments, performed in the absence of colipase and bile salts, support an absolute requirement of C-HPL for interfacial binding of HPL. To refine our analysis, we determined the contribution to lipid interactions of a hydrophobic loop (beta 5') in C-HPL by investigating a HPL mutant in which beta 5' loop hydrophobicity was increased by introducing the homologous lipoprotein lipase (LPL) beta 5' loop. This mutant (HPL-beta 5'LPL) penetrated into phospholipid monolayers at higher surface pressures than HPL, and its level of binding to TC8 was higher than that of HPL in the presence of serum albumin (BSA), an inhibitory protein that competes with HPL for interfacial adsorption. The beta 5' loop of LPL is therefore tailored for an optimal interaction with the surface of triglyceride-rich lipoproteins (VLDL and chylomicrons) containing phospholipids and apoproteins. These observations support a major contribution of the beta 5' loop in the interaction of LPL and HPL with their respective substrates.

The kinetics of the hydrolysis of 1,2-rac-dicaprin monomolecular films were measured from the decrease in the film area and the simultaneous changes in the surface potential at a constant surface pressure. A new kinetic model, based on the rate of product desorption is described. The surface potential data were interpreted in the light of the hydrolytic products accumulated at the interface. The surface concentration of the lipolytic products transiently present at the interface and the characteristic solubilization time of the lipolytic products were estimated. At surface pressures of more than 10 mN m−1, the amount of lipolytic products accumulated at the interface was found to be negligible in line with the simple kinetic model first developed in 1973 by R. Verger et al. (R. Verger, M.C.E. Mieras, G.H. de Haas, J. Biol. Chem., 248 (1973) 4023).

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.

Using the oil drop technique, we studied the effects of colipase and bile salts on the rate of hydrolysis of soybean oil by human pancreatic lipase (HPL) as well as on the interfacial binding. Upon continuously recording the decrease in the interfacial tension with time, a 10-15-fold increase in the HPL activity was found to occur in the presence of colipase. The catalytic rate constants of hydrolysis measured at the oil drop surface were found to be of the same order of magnitude as those obtained with monomolecular films spread at the air-water interface. Biotin-labeled HPL (HPL*) was used to determine the amount of adsorbed enzyme using an ELISA test. Less than 1% of the total amount of injected HPL* molecules was found to have adsorbed to the oil-water interface, and no significant effects of colipase on HPL* binding were observed. No significant changes in the hydrolysis rates or the binding of HPL* were detected in the presence of bile salts at concentrations ranging from below their critical micellar concentration (CMC) up to 100 microM. At the oil-water interface, in the absence or presence of bile salts below their CMC, it can be concluded that the colipase is a true lipase cofactor, i.e, it increases the enzyme turnover (approximately 10-15-fold) and does not affect the interfacial lipase adsorption.

Kinetic analyses of lipases and phospholipases must take into account the fact that these enzymes generally catalyze their reactions at a lipid-water interface as opposed to catalysis in a monophasic aqueous environment. This chapter discusses the application of an important tool in the kinetic modeling of these unique enzymes, the monolayer technique. The purpose of this chapter is to give an overview of kinetic modeling as applied to interfacial enzyme lipolysis, a brief discussion of the monolayer technique for studying lipase kinetics, and some applications of the monolayer technique to study substrate composition and stereoselectivity, lipolytic inhibitors, interfacial binding and acylglycerol synthesis.

Water-soluble lipolytic enzymes act mainly at the water/lipid interface where their catalytic reactions are coupled with various interfacial phenomena such as penetration and activation of the enzyme, as well as desorption of the soluble products, solubilization in the presence of accepters or molecular reorganization of the insoluble reaction products, inhibition, and so on. Various models have been proposed to analyze the kinetic data.

The desorption rates of monolayers of insoluble lipolytic products, oleic acid (OA) and mono-olein (MO), spread at the air-water interface, were measured in the presence of β-cyclodextrin (β-CD) or α-cyclodextrin (α-CD) at various subphase pH values. The desorption rates of the CD-OA complex at pH 2 can be satisfactorily described by a kinetic Langmuir equation revealing the existence of an energy barrier in the complex formation of the CD-OA. The dramatic increase observed in the desorption rates of the CD-OA complex at alkaline pH is in agreement with the theoretical prediction that a diffusion process is likely to occur from the surface to the bulk phase. Our results are consistent with the formation of hydrogen bonds between the ionized OA molecule and the O(6)H groups in the CD cavity. In the presence of β-CD in the subphase, comparable rates of hydrolysis of medium and long-chain monomolecular films of various glycerides by human pancreatic lipase (HPL) and Humicola lanuginosa lipase (HL) were observed. Comparisons between the above rates of enzymatic hydrolysis and the desorption rates of the CD-lipolytic product complexes indicated that the monomolecular film hydrolysis is rate limiting and does not depend on the type of CD used.

Surface pressure-molecular area curves of pure and mixed monolayers of lipolytic products (a mixture of oleic acid and 1,2-dioleoyl-sn-glycerol (OA-DO) at a 1:1 molar ratio), oleic acid (OA) and purified soybean oil (TG) spread at the argon-water interface were obtained. The results reveal that OA-TG and (OA-DO)-TG mixtures behave ideally when spread over a subphase at pH 8.0.We also determined the surface pressure-molecular area curves of lipolytic products (OA-DO) at the argon-water interface in the absence or presence of an excess of triglycerides. In the presence of an excess of TG, the films of OA-DO can be compressed up to surface pressures identical to the collapse pressure of the OA-DO when present alone. Furthermore, the fact that at the collapse pressure, the area occupied by the OA-DO mixture was the same in both the absence and presence of TG showed that under the experimental conditions used, negligible amounts of lipolytic products were dissolved in the oil and aqueous phases at both pH 5 and pH 8. The fraction of the surface occupied by OA-DO molecules varied from 80 to 100% at surface pressures ranging from 18 to 30 mN m(-1).

It has been known for some time that poly(dimethylsiloxanes) (PDMS) can form monolayers at the air-water interface. In this paper we show that formation of mixed monolayers of PDMS with fatty acids, di- and triglycerides is also possible. At the air-water interface silicone can interact and increase the retention of the fatty acids, which otherwise easily desorb into the aqueous subphase. Unlike fatty acids, long acyl chain tri- and diglycerides did not desorb into the aqueous subphase and their interaction with silicone was manifested by a condensing effect and a change in the collapse pressure of the mixed monolayers. Geotrichum candidum lipase (GCL) showed a similar level of binding to silicone monolayer as to an air-water interface. At low surface pressure (7.5 mN/m) the lipase hydrolyzed dicaprin in mixed 1,2-sn-dicaprin-PDMS monolayers at a rate 1.5-fold lower than the desorption rate of the capric acid from mixed monolayers of capric acid and PDMS. Compression of the dicaprin-PDMS monolayer to 20 mN/m led to a 15-fold increase in lipase activity (70% of the activity observed for monolayer of pure dicaprin compressed to the same surface pressure), indicating an increase in dicaprin's surface concentration and its accessibility to the enzyme. The lipase activity profile determined titrimetrically against varying amounts of glycerides in mixed glyceride-PDMS emulsions (at constant 5% oil-phase concentration) resembles the lipase activity-surface pressure profile obtained with the monolayer method when using pure glycerides. This suggests that the location of glycerides in condensed mixed monolayers with PDMS should be similar to that in mixed glyceride-PDMS emulsions. By changing the molar fraction of tristearin and tripalmitin in the mixture with PDMS, it was possible to regulate the quality of the interface in emulsions and to optimize the enzymatic hydrolysis of these solid triglycerides.

The desorption rates of monomolecular films of oleic acid (OA) and lysophosphatidylcholine (lyse PC) at the air/water interface by water soluble beta-cyclodextrin (beta-CD) were studied. The desorption of OA and lyse PC involves the complexation of the single acyl chain into the beta-CD cavity and the solubilization of the beta-CD/OA or beta-CD/lyso PC complex into the aqueous subphase, associated with a rapid decrease in the surface pressure. In the case of monomolecular films of egg phosphatidylcholine (egg PC), 1,2-dihexadecanoyl-sn-glycero-3-phosphatidylcholine (DPPC); 1,2-di(cis-9-octadecenoyl)-sn-glycero-3-phosphatidycholine (DOPC), no detectable changes in the surface pressure occurred after beta-CD injection into the subphase.The hydrolysis of medium and long chain PC monomolecular films by phospholipase A(2) (PLA(2)) was investigated in the absence of beta-CD in the subphase. The considerable difference between the apparent kinetic constants was attributed to physical steps possibly involving molecular reorganization in the interface of long chain lipolytic products, associated with enzyme product inhibition. In the presence of beta-CD in the subphase, the enzymatic hydrolysis of monomolecular films of long chain PC was found to have kinetic constants which were comparable to those measured with medium chain lipids.Furthermore, comparisons between the desorption rates of the beta-CD/lipolytic product complexes and the enzymatic hydrolysis rates of long chain PC, in the presence of beta-CD in the aqueous subphase, showed that the rate limiting step is neither the formation of the beta-CD/lipolytic product complexes nor their desorption into the water subphase but the hydrolysis of the PC monomolecular films by PLA(2).The presence of beta-CD in the water subphase made it possible for the first time to perform kinetic measurements on the rates of hydrolysis of monomolecular films of long chain PC by PLA(2).

A versatile and continuous assay for phospholipase D (PL D) activity was developed using the monomolecular film technique. For this purpose, a two-step enzymatic reaction was used. First, PL D hydrolysis of stable 1,2-diacyl-sn-glycero-3-phosphocholine (PC) films by PL D generated a stable 1,2-diacyl-sn-glycero-3-phosphate (PA) film and water-soluble choline. Secondly, the latter acidic phospholipid, in contrast to the initial PC molecule, was further hydrolysed under the action of porcine pancreatic lipase (PPL) in order to give rise to lysophosphatidic acid and fatty acid, which were rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the PL D-catalyzed reaction with phospholipid monolayers as substrates. The PL D kinetics were linear with time and the velocities recorded were directly dependent upon the amount of PL D used. In a preliminary study, we investigated the effects of the surface pressure on the PL D activity.

The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.

The kinetics of surface film formation from Intralipid(TM) 20% spread at the air-water interface were studied by recording the variations with time of the surface pressure and surface potential. A simple kinetic scheme describing the interfacial destabilization of both emulsion and liposomes particles was developed. The rate constants of the surface transformation of both emulsion and liposomes particles into a mixed triolein-egg phosphatidylcholine (PC) monolayer were determined and compared with the rate constant of the surface transformation of liposomes. The higher rate constant of spreading observed with Intralipid(TM) was attributed to the higher spreading capacity of the core of the emulsion particles. The rate constant of destabilization of the PC monolayer covering the emulsion particles was nearly twice as high as that of the PC bilayer of liposomes.It was observed that the surface transformation of Intralipid(TM) emulsions were accelerated by enzymatic hydrolysis when human gastric lipase and human pancreatic lipase were introduced. It was further confirmed that preincubating the Intralipid(TM) with HGL and HPL consecutively accelerated the Intralipid destabilization.