Enzyme--substrate interaction in lipid monolayers. III. A study of the variation of the surface concentration with lipolysis.

Monolayers of a diacylglycerol were submitted to the action of lipase, keeping the area constant. The variation of lipase, keeping the area constant. The variation of the surface concentration gamma of the substrate with time was derived from the recorded reduction of the surface pressure pi (the isotherm of the monolayer being previously established). The rate -d gamma/dt was determined both as a function of the surface concentration gamma of the substrate and as a function of the bulk concentration C of the enzyme in the underlying solution. The rate depends on the quantity of enzyme ze adsorbed on the monolayer and on the enzymatic specific activity alpha of these adsorbed enzyme molecules. Both ze and alpha vary with gamma. The two variations have been quantitatively dissociated. The curves of ze and of alpha as functions of gamma coincide with those previously established in the study of hydrolysis under constant surface pressure.     

Effect of diolein on hydrolysis of phosphatidylcholine by phospholipase C from Clostridium perfringens.

The activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.     

Enzyme-substrate interaction in lipid monolayers. II. Binding and activity of lipase in relation to enzyme and substrate concentration and to other factors.

With the limited stirring procedure used in the present work, substrate and enzyme together form a segregated and well-defined system on the surface. The lipase molecules responsible for the lipolysis are only those that are adsorbed on the glyceride monolayer. After a study of the stirring procedure, two series of systematic experiments were done: a) the bulk concentration of the enzyme was varied with different constant surface concentrations of the substrate, and b) the surface concentration of the substrate was varied with different constant bulk concentrations of the enzyme. The influence of the surface concentration of substrate on a) the rate of lipolysis, V,; b) the enzyme activity, a,; and c) the enzyme adsorption, Ze, were each determined by a different procedure. The values obtained verify the enzymic activity equation (a = V/Ze). The roles of other factors (Ca2+ ions, and pH) which govern the adsorption of the enzyme and its specific activity were also studied in preliminary experiments.     

Adsorption properties and activities of lipase on a gold substrate modified by self-assembled monolayers.

The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.     

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.     

Adsorption Properties and Activities of Lipase on a Gold Substrate Modified by Self-assembled Monolayers

The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.     

Lateral lipid distribution is a major regulator of lipase activity. Implications for lipid-mediated signal transduction.

Pancreatic carboxylester lipase catalyzes the exchange of 18O between water and 13,16-cis,cis-doco-sadienoic acid (DA) in monolayers at the argon-buffer interface (Muderhwa, J.M., Schmid, P.C., and Brockman, H.L. (1992) Biochemistry 31, 141). In mixed monolayers of 18O, 18O-DA and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), both the extent and mechanism of 18O exchange show characteristics of a critical transition in the range of 0.5-0.6 mol fraction of DA (Muderhwa, J.M., and Brockman, H. L. (1992) Biochemistry 31, 149). To determine if the regulatory behavior exhibited on this type of surface is limited to members of the carboxylester lipase gene family (cholinesterases), comparable experiments were performed with a genetically and functionally unrelated lipase, pancreatic colipase-dependent lipase (PL). PL readily catalyzed the exchange of 18O between water and the carboxyl group of DA with enzyme at either monolayer or catalytic levels in the fatty acid-buffer interface. The oxygen exchange reaction obeyed a random, sequential mechanism, indicating that the dissociation of the enzyme.DA complex is much faster than the rate-limiting step in the overall exchange process. Kinetic analysis of oxygen exchange in pure DA monolayers showed a first-order dependence on interfacial PL and DA concentrations from which kcat/Km values were calculated. The oxygen exchange reaction proceeded with a rate constant of 16 x 10(-2) cm2 pmol-1 s-1, a value comparable to that for hydrolysis of the ester substrate, 1,3-dioleoylglycerol. With a monolayer of PL adsorbed to the interfacial phase, kcat/Km for oxygen exchange was about 600-fold lower than the value obtained with catalytic levels of adsorbed enzyme, indicating a possible restriction of substrate diffusion in the protein-covered fatty acid monolayer. With constant bulk PL concentration and mixed lipid monolayers containing DA and the non-substrate lipid, POPC, the extent of oxygen exchange increased abruptly as the abundance of DA in the interface was increased from 0.5 to 0.6 mol fraction. Concomitant with this critical transition was a change in the apparent mechanism of oxygen exchange from coupled to random, sequential. For both the extent of oxygen exchange and its mechanism shift, the critical transition was independent of the lipid packing density, i.e. surface pressure, of the interface. These results show that PL responds similarly to carboxylester lipase with respect to changes in interfacial lipid mole fraction in DA-POPC surfaces.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of carboxylester lipase adsorption to surfaces. 1. Chemical specificity

The chemical specificity of the adsorption of porcine pancreatic carboxyl ester lipase to pure lipid surfaces was examined. Adsorption of native and catalytically inactivated enzyme was measured at the argon-buffer interface by using lipid films near the point of collapse. Protein adsorbed readily to films of triolein, 1,3-diolein, methyl oleate, oleonitrile, oleyl alcohol, and 13,16-docosadienoic acid. However, recovery of enzyme activity was variable. These differences and the changes in surface pressure accompanying adsorption indicated the occurrence of enzyme denaturation at the interface. Denaturation was controlled largely by surface free energy but showed some chemical specificity at high surface pressures. Adsorption of protein to the lipids was comparable when measured under either equilibrium or initial rate conditions. Together with surface pressure changes that accompany adsorption, the data indicate a relative lack of specificity for the enzyme-surface interaction. Adsorption to 13,16-docosadienoic acid and 1,3-diolein obeyed the Langmuir adsorption isotherm. Dissociation constants ranged from 10 to 50 nM, depending on enzyme form, ionic strength, and pH. With both lipids, a monolayer of enzyme was adsorbed at saturation. In contrast to these results, adsorption of enzyme activity and protein to films of 1-palmitoyl-2-oleoyl-phosphatidylcholine was less than or equal to 5% of that observed with the other lipids under all conditions. Comparison of rate constants for adsorption to 13,16-docosadienoic and 1,3-diolein as a function of subphase pH indicated a marked dependence on the ionization state of the fatty acid. Overall, the data suggest that the presence of zwitterionic and anionic lipids may regulate the interaction of the enzyme with substrate-containing surfaces in vivo.     

Surface specific kinetics of lipid vesicle adsorption measured with a quartz crystal microbalance.

We have measured the kinetics of adsorption of small (12.5-nm radius) unilamellar vesicles onto SiO2, oxidized gold, and a self-assembled monolayer of methyl-terminated thiols, using a quartz crystal microbalance (QCM). Simultaneous measurements of the shift in resonant frequency and the change in energy dissipation as a function of time provide a simple way of characterizing the adsorption process. The measured parameters correspond, respectively, to adsorbed mass and to the mechanical properties of the adsorbed layer as it is formed. The adsorption kinetics are surface specific; different surfaces cause monolayer, bilayer, and intact vesicle adsorption. The formation of a lipid bilayer on SiO2 is a two-phase process in which adsorption of a layer of intact vesicles precedes the formation of the bilayer. This is, to our knowledge, the first direct evidence of intact vesicles as a precursor to bilayer formation on a planar substrate. On an oxidized gold surface, the vesicles adsorb intact. The intact adsorption of such small vesicles has not previously been demonstrated. Based on these results, we discuss the capacity of QCM measurements to provide information about the kinetics of formation and the properties of adsorbed layers.     

Hydrolysis of mixed monomolecular films of triglyceride/lecithin by pancreatic lipase.

The main purpose of this study was to describe the influence of lecithin upon lipolysis of mixed monomolecular films of trioctanoylglycerol/didodecanoylphosphatidycholine by pancreatic lipase in order to mimic some physiological situations. The quantity of enzyme adsorbed to the interface was simultaneously determined using 5-thio-2-nitro[14C]benzoyl lipase. Lipolytic activity was enhanced 3- to 4-fold in the presence of colipase, an effect which is attributed to increased enzyme turnover number. When a pure triglyceride film was progressively diluted with lecithin, the minimum specific activity of lipase exhibited a bell-shaped curve: a mixed film containing only 20% trioctanoylglycerol was hydrolyzed at the same rate as a monolayer of pure triglyceride.     

A spectrophotometric assay for lipase activity utilizing immobilized triacylglycerols.

New substrates for the determination of lipase activity have been developed. Triacylglycerols were immobilized by adsorption on an appropriate carrier or adsorbent yielding a lipase substrate in a powder form. The adsorbed triacylglycerols were easily hydrolyzed by lipases present in a reaction mixture. The released fatty acids were extracted with benzene and converted to the corresponding Cu (II) salts (copper soaps) which were measured spectrophotometrically.     

Effect of Different Supports on the Reaction Rate of Rhizomucor Miehei Lipase in Organic Media

Five different aluminas, a silica and a zirconia support were used to adsorb lipase (E.C. 3.1.1.3) from Rhizomucor miehei. The activity of the immobilised lipase was measured by esterification of dodecanol and decanoic acid in hexane. The immobilised lipase and the organic phase were pre-equilibrated separately to known water activities before mixing them to commence the reactions. The aluminas, which varied in pore sizes and surface areas, adsorbed similar amounts of enzyme. However, the esterification activities varied about 10-fold, increasing with increasing surface area. The silica and zirconia supports adsorbed about half as much lipase as the aluminas. The esterification reaction rates per unit quantity of enzyme adsorbed were compared with those for aluminas with similar surface areas; this specific rate was about 2 times higher for the zirconia, but the difference with silica was only small. There was no clear correlation between the esterification rates at fixed water activity and the amount of water adsorbed by the support used.     

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.     

Studies on the mechanism of the lipase reaction. II. Comparative studies on the adsorption of lipases and various proteins at the air-water interface.

Adsorption of lipases (EC 3.1.1.3) and various proteins at the air-water interface has been investigated in relation to the mechanism of lipase reaction. Aqueous solutions of lipases and denaturated proteins show surface activity as strong as that of synthetic detergents. However, ths surface activity of esterases and various other proteins is little or none. By foam fractionation it was shown that lipases were adsorbed at the air-water interface and the adsorption followed the equation of Langmuir's adsorption isotherm. The properties of lipase at the interface are discussed in relation to the mechanism of lipase reaction and the differences from the esterase reaction.     

Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., gold modified with a methyl-terminated, self-assembled alkylthiol layer. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance (QCM). Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy (AFM). Lipase adsorption from low ionic strength aqueous buffer produced a uniform confluent protein monolayer. Inclusion of 10% (vol) ethanol in the buffer during immobilization resulted in a 33% adsorbed mass increase. Chemically similar cosolvents, all at 10% by volume in buffer, were also individually examined for their influence on CALB adsorption. Glycerol or 1-propanol increased mass adsorption by 10%, while 2-propanol increased mass adsorption by 33%. QCM dissipation values increased threefold with the inclusion of either ethanol or 2-propanol in the medium during lipase adsorption, indicating formation of multilayers of CALB. Partial multilayer formation using 10% ethanol was confirmed by AFM. Inclusion of 10% ethanol in the CALB immobilization buffer decreased the specific activity of the immobilized lipase by 37%. The formation of lipase multilayers in the presence of certain cosolvents thus results in lower specific activity, which might be due to either influences on lipase conformation or substrate active site accessibility.     

Yeast alcohol dehydrogenase bound to membranes: surface and microenvironment effects on activity and stability.

The enzyme, yeast alcohol dehydrogenase, was adsorbed to porous nitrocellulose and nylon membranes. The two membranes provide different surface chemistries as indicated by the results of the streaming potential, enzyme adsorption, and fluorescein isothiocyanate adsorption experiments. The stability of the enzyme, as determined by continually measuring the extent of coenzyme reduction as a function of time, appeared to be much less for the enzyme adsorbed to the positively charged membrane surface. Moreover, the enzyme adsorbed to the positively charged membrane was the least responsive to pulses of the reducing agent, dithiothreitol, and appeared to exhibit the highest transition temperature when subjected to differential scanning calorimetry analysis. These results indicate that the entropically spreading process observed for other adsorbed proteins may be occurring and the process is more rapid and extensive when enzyme is adsorbed to the nylon than the nitrocellulose membrane. In addition to the relative stability of the enzyme on two different surfaces being examined, the effect of the microenvironment on modulating the activity of the enzyme was investigated by using the reversibility of the enzyme-catalyzed reaction as a probe of the average local environment of the enzyme. It was found that a threshold buffer concentration existed that, once exceeded, the effect of proton production by the reaction could be suppressed.     

Saccharification and adsorption characteristics of modified cellulases with hydrophilic/hydrophobic copolymers.

Saccharification and adsorption characteristics of native and modified cellulases were investigated. Copolymers, containing polyoxyalkylene and maleic anhydride (MA) were used to modify cellulase. Amino groups of the cellulase were covalently coupled with the MA. As the degree of modification (DM) increased, the activity of modified cellulase slightly decreased. At the maximum DM, the modified cellulase activity retained more than 75% of the unmodified native cellulase activity. In saccharification, native cellulase rapidly adsorbed onto the substrate at initial reaction time. Native cellulase adsorbed tightly onto the substrate surface and did not desorb as reaction time proceeded. The strong adsorption of cellulase onto the substrate can, however, be controlled by the modification. As the hydrophilicity of modified cellulase increased, free modified enzyme concentration also increased. As a result, the conversion rate of modified cellulase was higher than the native one.     

Lipase adsorption as a factor of lipolysis regulation

Sorption isotherms of pancreatic lipase on solid supports were studied. It was shown that the enzyme adsorption can be described by Langmuir equation for hydrophobic surface and by the equation which takes into account reversible dimerization of the protein in the absorption layer for hydrophilic surface. The catalytic properties of adsorbed lipase depend on the nature of solid support. The significant role of the structure of adsorption layer in heterogeneous activation of the enzyme on hydrophobic surface was suggested.     

Hydrolysis of lipid monolayers and the substrate specificity of hepatic lipase

The substrate specificities of the phospholipase and triglyceridase activities of purified rat liver hepatic lipase were compared using lipid monolayers so that the substrates were presented to the enzyme in a controlled physical state. The rate of hydrolysis of 14C-labeled lipid at constant surface pressure in the presence of hepatic lipase and fatty acid-free bovine serum albumin at 33 degrees C was determined by monitoring the decrease of surface radioactivity. In monolayers of sphingomyelin/cholesterol (2:1, mol/mol) containing either 1 mol% triacylglycerol, 1 mol% phosphatidylethanolamine, or 10 and 20 mol% phosphatidylcholine, hepatic lipase clearly showed a preference for unsaturated over saturated lipids. In addition, with a sphingomyelin/cholesterol (2:1) monolayer containing 1 mol% of lipid substrate, hepatic lipase showed the following preference: triolein = dioleoylphosphatidylethanolamine much greater than dioleoylphosphatidylcholine; the respective rates of hydrolysis were 15.3 +/- 1.2, 14.9 +/- 0.8, and 0.5 +/- 0.1 mumol fatty acid produced/h per mg hepatic lipase. Overall, it appears that when comparing rates of hydrolysis of molecules within a given lipid class, hydrocarbon chain interactions are important. However, when comparing different lipid classes such as phosphatidylcholines and phosphatidylethanolamines, it is apparent that the polar group has a significant influence on the rate of hydrolysis. The rate of [14C]triolein hydrolysis, when mixed at surface concentrations of up to 2 mol% in a sphingomyelin/cholesterol (2:1) monolayer, was significantly faster than when triolein was present in a 1-oleyl-2-palmitylphosphatidylcholine monolayer; the rates of hydrolysis were 47.7 +/- 5.4 and 8.9 +/- 0.8 mumol fatty acid produced/h per mg hepatic lipase, respectively. The monolayer physical state and the miscibility of the substrate in the inert matrix influence the presentation of the substrate to the enzyme, thereby affecting the hydrolysis rate.     

Dynamic adsorption behavior of poly(3-hydroxybutyrate) depolymerase onto polyester surface investigated by QCM and AFM

Time-dependent adsorption behavior of poly(3-hydroxybutyrate) (PHB) depolymerase from Ralstonia pickettiiT1 on a polyester surface was studied by complementary techniques of quarts crystal microbalance (QCM) and atomic force microscopy (AFM). Amorphous poly(l-lactide) (PLLA) thin films were used as adsorption substrates. Effects of enzyme concentration on adsorption onto the PLLA surface were determined time-dependently by QCM. Adsorption of PHB depolymerase took place immediately after replacement of the buffer solutions with the enzyme solutions in the cell, followed by a gradual increase in the amount over 30 min. The amount of PHB depolymerase molecules adsorbed on the surface of amorphous PLLA thin films increased with an increase in the enzyme concentration. Time-dependent AFM observation of enzyme molecules was performed during the adsorption of PHB depolymerase. The phase response of the AFM signal revealed that the nature of the PLLA surface around the PHB depolymerase molecule was changed due to the adsorption function of the enzyme and that PHB depolymerase adsorbed onto the PLLA surface as a monolayer at a lower enzyme concentration. The number of PHB depolymerase molecules on the PLLA surface depended on the enzyme concentration and adsorption time. In addition, the height of the adsorbed enzyme was found to increase with time when the PLLA surface was crowded with the enzymes. In the case of higher enzyme concentrations, multilayered PHB depolymerases were observed on the PLLA thin film. These QCM and AFM results indicate that two-step adsorption of PHB depolymerase occurs on the amorphous PLLA thin film. First, adsorption of PHB depolymerase molecules takes place through the characteristic interaction between the binding domain of PHB depolymerase and the free surface of an amorphous PLLA thin film. As the adsorption proceeded, the surface region of the thin film was almost covered with the enzyme, which was accompanied by morphological changes. Second, the hydrophobic interactions among the enzymes in the adlayer and the solution become more dominant to stack as a second layer.