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LIPASE KINETICS AT THE TRIACYLGLYCEROL-WATER INTERFACEA new method,-called the "oil-drop method", was developed and adapted to studing the rate of enzymatic reactions, using long chain triacylglycerols, the main physiological substrates of digestive lipases. The method is based on the variations versus time in theoil/waterinterfacial tension (Yo/w) resulting from the accumulation of water insoluble lipolytic products on the surface of a drop (1). Measurements were carried out with pure Porcine Pancreatic Lipase (PPL). This method was also used to detect Human Gastric Lipase (HGL)at low pH,since difficulties were encountered in earlier studies when measuring lipolytic activity under acidic conditions (2). The lipolytic kinetics under high hydrostatic pressure (800 and 1200 bars) were also investigated with the oil-drop method, and a specific two-fold increase in lipase activity was found to have occured. A newprototype is being developed for automatically analyzing the oil-drop profile is being developed in order to improve the data acquisition rate andthe accuracy of the measurements.
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STEREOSELECTIVITY OF LIPASES: Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film techniqueIn the present study, porcine pancreatic lipase (PPL), rabbit gastric lipase (RGL) and humangastric lipase (HGL) stereospecificity towards enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1 ,2)-diacyl-sn-glycerol or enantiomeric 1(3)-alkyl-2-acyl-snglycerol or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the samein both enantiomers, thereby excluding the possibility of effects occuring dueto fatty acid specificity. We observed a PPL sn-3 stereoselectivity when using the enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack ofstereoselectivity observed when using the enantiomeric 1(3)-alkyl- 2,3(1,2)-diacyl-sn-glycerol (triglyceride analogues). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyse the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoalky] diacylpairtested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of RGL, whereas low rates and a clear chiral discrimination was noticed in the case of HGL duringcatalysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers.It is particulary obviousthat in the case of HGL decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylaminoderivatives (diglyceride analogues).
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ENZYMEKINETICS OF LIPOLYSIS: Lipase inhibition by proteinsApart from their general biological significance, lipolytic enzymes play an increasingly important role in biotechnology: clinical medicine, pharmacology, nutrition, food andoil technology. Lipids constitute a large part of the biomassof the hearth andlipolytic enzymes play an essential role in the metabolic turnoverofthese lipids. Phospholipids and glycolipids constitute fundamental and universal structural elements of biological membranes. Onthe other hand, triglycerides constitute the main energy reservoir of higher animals. Lipases are required in the lipid transfer from one organism to another. Forthe biochemist, perhaps the most importantand fascinating aspectof lipolytic enzymesis the unique physicochemical character ofthe reactions they catalyze. These enzymesare perfectly water soluble andact very efficiently on water insoluble lipidic substrates which spontaneously self organize in water as monomolecular films, bilayers, liposomes, emulsions or micelles. This catalysis is essentially occurring at the lipid/waterinterface. Conventional enzymekineticsis oflittle relevance under these heterogeneous conditions. The development ofan "interfacial enzymology" would be of general interest forall biological sciences because in nature most enzymatic reactions take place at membranes. Thereare at least 3 major reasonsfor using lipid monolayers as substratesfor lipolytic enzymes: 1. The technique is highly sensitive and very little lipid is needed to obtain kinetic measurements. 2. A rather common observation reported by many authors working onthekinetics oflipolytic enzymesis the presence of a lag period in the hydrolysis of both emulsions, liposomes, micelles and monolayers. Such studies should preferably be done on monolayers ofshort-chain lipids where the perturbing influence of increasing amounts of reaction products can be minimized. 3. Probably the most important reason fundamentally is the possibility of varying the "quality of interface" determined by the nature ofthe lipids forming the monolayer, the orientation of the molecules, molecular and charge density, water structure, fluidity, etc...
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PHOSPHOLIPID-GLYCERIDE INTERACTIONS AS REGULATORS OF CARBOXYLESTER LIPASE ADSORPTION AND CATALYSISLipolysis occurs in the presence of non-substrate, surface-active molecules like phosphatidylcholines (PC’s). The role of PC in the regulation of carboxylester lipase (CEL) was investigated using mixed lipid monolayers. For each substrate an abrupt increase in substrate hydrolysis from <10% to >90%, i.e. switching, occurred at a substratedependent lipid composition. Similar results were obtained for free fatty acid 180 exchange catalyzed by CEL. The lack of hydrolysis at low substrate was not due to an absence of CEL at the interface. Enzyme adsorption to substrate.PC films is consistent with all surface being available, except for 43.5 AZ /molecule of PC. Adsorption of CEL obtained by extrapolation to infinite dilution of PC was 0.92 pmol/cm?, 1/4 of the expected value. CEL adsorption to PCefatty acid films fell short of the values predicted from substrate-containing films. These results suggest that adsorption of CEL to PC-rich films is regulated by clusters or domains of substrate or fatty acid molecules in the PC interface. Such clusters are predicted from stochastic distribution of components but appear to be modulated by lipid-lipid interactions. The stochastic argument further predicts that substrate domains should interconnect if >45% of total surface area is non-excluded by PC. Using data obtained from CEL binding to calculate non-excluded area at each lipid composition, hydrolysis and 180-exchange data are consistent with catalysis being regulated by domain connectivity, i.e. percolation. Being lipid-based, this regulation should occur with other lipases, but the consequences of the organization may be lipase specific.
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CATALYTIC MECHANISM OF PHOSPHOLIPASE A2Phospholipases A2 (MW 14,000) specifically hydrolyse the 2-acyl linkage of phosphoglycerides in a calcium dependent reaction. From the three-dimensional structure of native bovine pancreatic phospholipase Az at 1.7 A resolution in conjunction with biochemical evidence the architecture of the active site became apparent and a mechanism for the hydrolysis of both monomeric as well as aggregated phospholipids could be proposed. In this mechanism a water molecule activated by His48 was suggested as the nucleophile. The enzyme possesses an extended area around the entrance to the active site that is involved in the binding of aggregated substrates. Pro-phospholipase can not degrade these aggregated substrates and it appears that in pro-phospholipase part of this binding area is flexible. The three-dimensional structure of a phospholipase Az mutant complexed with a substrate analogue shows that the substrate analogue is bound to the calcium ion in the enzyme’s active site both by its phosphate group and by the carbonyl oxygen of the fatty acid residue bound at the C2 atom of the glycerol backbone. The sn-2 chain of the inhibitor makes extensive hydrophobic contacts with the disulfide bond between residues 29 and 45, and the side chains of residues Leu2, Phe5, lle9, Leu19, Phe22 and Tyr52. The hydroxyl group of Tyr69 is hydrogenbonded to one of the oxygen atoms of the inhibitor’s phosphate group.
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CRYSTALLISATION AND PRELIMINARY CRYSTALLOGRAPHIC STUDIES OF LIPASESLipases from two Pseudomonas species have been crystallised in various forms some of which appear suitable for a detailed structural analysis by X-ray crystallography. All crystals were grown using the method of vapour diffusion by hanging drop or sitting drop, at 4C or 15C. Different crystal forms tended to appear at the two temperatures chosen for the crystallisation trials. The two lipases used came from Pseudomonas Amano, and Pseudomonas Glumae. Crystals appeared under different conditions for the two enzymes, but at least one crystal form of each diffracted to greater than 3.0A. The work on P. Amano has as yet gone no further than the ascertation of conditions for crystallisation to occur, but space groups have been determined for three crystal forms of P. Glumae. The first crystals that were tested proved to be trigonal, but were unsuitable for detailed investigation, the two others were both possible candidates for high resolution studies. One form is tetragonal, crystallising in space group P422, the other is orthorhombic, the crystals growing in space group P222, It is on this second form that subsequent structural analysis has been concentrated.
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CRYSTALLIZATION AND PRELIMINARY X-RAY ANALYSIS OF A LIPASE FROM A SPECIES OF PSEUDOMONASA lipase from a species of Pseudomonas has been cloned and expressed in E. coli. Variants of this lipase have been generated, using sitespecific mutagenesis, that have significantly altered k,,,, K, and substrate specificity. We have undertaken to determine the threedimensional structure of this enzyme using X-ray crystallography. Crystals have been obtained and these crystals diffract to 2.5 Angstrom resolution. We are now in the process of evaluating heavy atom derivatives to be used to improve the phase information used to calculate electron density maps.
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CRYSTALLOGRAPHIC STUDY OF A RECOMBINANT CUTINASE FROM FUSARIUM SOLANIPISICutinases are a group of extracellular fungal hydrolytic enzymes capable of degrading the insoluble lipid polyester matrix,i.e. cutin, which covers the surface of plants. Their weight is 22,000 Da, signifantly lower than all other lipases. A recombinant cutinase from F. solani pisi is expressed and excreted with very high yieldsin E. coli cultures. Cutinase wascrystallized (PEG 6000 15-20% ,pH 7.0 to 10.0,20°C) in space group P21 with cell dimensions 35.1A,67.4A,37.05 A, ß=94°.They diffract to 1.5 A resolution (Rsym=4.41%). Data from native and derivatives have been collected. MIR phasingis in progress.
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CRYSTALLIZATION AND CHARACTERIZATION OF CANDIDA RUGOSA LIPASEA lipase isolated from the fungus Candida rugosahas beenpurified and crystallized in a form suitable for X-ray crystallographic structure determination. Several proteins with lipolytic activity having isoelectric points from 4.2 to 5.8 were separated by ion exchange chromatography.The protein having the lowestisoelectric point was crystallized from 2-methy]-2,4-pentandiol in MES bufferin the presenceof calcium (II) salts. The crystals with cell dimensions a=64.9(1) 7 b=97.2(1) A and b=175.8(2)A, grow as large colorless plates often exceeding 0.7 mm in each of two dimensions. From the diffraction pattern the apparent crystal symmetry is C222,. Experimental density determination suggests one 60,000 M,. molecule in the asymmetric unit and approximately 50% solvent by volume. Theratio unit cell volume to the molecular weightof the contents of the unit cell, Vj, is 2.3 A3/d. Diffraction is strong to a resolution of 2 A resolution and work is underway to determine the three dimensional structure of this enzyme.
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Crystallization and Preliminary X-ray Studies of Lipase from Geotrichum candidumWehave succeeded in the production of new crystals of lipase from Geotrichum candidum which were suitable for a refined X-ray analysis. First measurements proved the lattice constant and space group to be similar to that reported by Hata et al. /1/. However, these crystals can be analyzed without any cross-linking. This should enable a higher resolution for the structure determination of the lipase from Geotrichum candidum. Inhibition experiments with this lipase proved metal ions, iodine and p-chloromercuribenzoate to effect the enzyme activity. Metal ions inactivated lipase at a 1000-fold excess and in the order of Ag*>Hg’* >Co**>Zn**. In contrast, I, and p-chloromercuribenzoate modified lipase from Geotrichum candidum in stoichiometric amounts. These modified as well as a deglycosylated lipase could becrystallized. Such crystals ofslightly modified lipases offer the chance of analyzing further details of the lipase structure, e.g. the influence of the glycosylation on the tertiary structure, and they are an alternative approach to high quality crystals.
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STRUCTURAL STUDIES OF GEOTRICHUM CANDIDUM LIPASELipase from Geotrichum candidum has been purified to homogeneity and crystallized in three different forms. Rotation function analysis indicates close relationship in molecular packing in all forms. Despite intensive search only one isomorphous heavy atom derivative has been found to date. A further search is in progress.
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X-RAY STRUCTURE ANALYSIS OF GEOTRICHUM CANDIDUM LIPASE AT 2.8 A RESOLUTIONGeotrichum candidum (ATCC 34614) produces extracellular lipase upon submerged cultivation. With conventional purification techniques, the enzyme was easily purified to apparent homogeneity, as judged from disc- PAGE, isoelectric focusing and ultracentrifiugation. It was easily crystallized by concentration, and low resolution X-ray analysis was done on the crystals. However, we have quite recently found that the apparently pure lipase was a mixture of two isozymes, both composed of 544 amino acids and 6.5% carbohydrate. The two isozymes were separated from each other by hydrophobic interaction chromatography. An X-ray analysis at 2.8 A resolution has been undertaken onthe main fraction. The main chain was tentatively followed. The whole enzyme molecule consists of a single domain of 50 x 50 x 70 A, and contains 9 a-helices and 2 g-sheets.
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THE CRYSTAL STRUCTURE OF LIPASE FROM MUCOR MIEHEIThe crystal structure of lipase from the fungus Mucor miehei has been determined; it has revealed the enzyme's main chain structure as well as the details of the interactions madeby the individual sidechains. The enzymecontains a central 8 strand 6 sheet structure that extends acrossthe full depth of the molecule. Arranged across this, in some of the segmentslinking the strands, are several helices which pack against the sheet structure. There is an N terminal helix which appearsto sit at the centre of the convex surface created by the ß sheet. The serine (144) at the catalytic site has ben identified by chemical experiment. Inspection of the structure at this serine showedit to be part ofa triad: asp... his ... ser, equivalent at the active atomsto that seen in the serine proteases. Thereis no similarity in the lipase main chain structure to those of the trypsin related or the subtilisin related serine proteases - thus the appearanceofthe asp- his - ser triad is an example of an independentsolution of these side chainsfor a catalytic reaction. There is a small helix situated over the catalytic residues, effectively blocking them from the surrounding solvent. This lid explains the inactivity of the enzyme in aqueous conditions. The side chains on this helix are on one side polar and on the other nonpolar. This suggests that underthe influence of the interface at a micelle the lid could be destabilised by non-polar interactions andbe displaced, exposing the catalytic triad to the lipid at the interface.
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PANCREATIC LIPASE : CRYSTALLIZATION, DOMAIN INVESTIGATION AND CLONINGPancreatic lipase is involved in dietary fat digestion. The enzyme, which belongs to a special class of esterases, realizes an heterogeneous catalysis. Although extensively studied for many years, the mechanism of action of lipase on emulsified substrates is still not elucidated. Crystals of man and horse pancreatic lipases suitable for an X-ray investigation have been obtained. The cell parameters of the various crystalline forms have been determined. Heavy atom derivatives are under investigation. The spatial organization of pancreatic lipase in two well defined domains has been studied through limited proteolysis of the enzyme from various species. A horse pancreatic cDNA library has been built and the cDNAs encoding for lipase and colipases isolated.
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CRYSTALLOGRAPHIC STUDIES OF THE PANCREATIC LIPASE/COLIPASE SYSTEMCrystals of a complex between porcine pancreatic lipase (50000 Da) andits cofactor, colipase (11000 Da), have been obtained. These 1/1 complex crystals are disordered, and diffract weakly. Horse pancreatic lipase has been crystallized in space group P21242), (89A,97A,145A ; pH 6.0, PEG 8000 10%, 20°C) and a 2.3 A native data set has been collected. One PCMBS derivative was not sufficient to produce a good map,andwearestill looking for other derivatives. Two crystal forms of human pancreatic lipase (purified from pancreas juice) have been obtained, different from the one reportedin thelitterature. They crystallize in the same conditions and space groupsare P2, and P1. Native data sets have beencollected.
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CRYSTALLIZATION OF GASTRIC LIPASESGastric lipases are pH resistant enzymes with a molecular weight of ca 50,000, including 15% sugars. Every glyco-variant from different species have been purified by ief, and submitted to crystallization, using parameters found by an incomplete factorial analysis method. All attempts were successful, but most crystals were unsuitable to X-ray studies due to huge cell dimensions. Dog gastric lipase crystals, the most suitable for X-ray study (pH 6.8, 12% PEG 6000, 20°C; P2242 :182A, 211A, 98 A), contain ca 8 molecules in the asymetric unit , and therefore diffract to only 4A on lab sources.
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CRYSTALLIZATION AND X-RAY STUDIES OF LIPASESSeveral structure determinations of lipases are underway in Marseille. Most projects are at a heavy-atom derivative search stage. These projects belong to three classes: i. Cutinases are a group of extracellular fungal hydrolytic enzymes capable of degrading the insoluble lipid polyester matrix, i.e. cutin, which covers the surface ofplants. Their weight is 22,000 Da, signifantly lower than all other lipases. A recombinant cutinase from F. solani pisi is expressed and excreted with very high yields in E.coli cultures. Cutinase was crystallized (PEG 6000 15-20% ‚pH 7.0 to 10.0,20°C) in space group P2, with cell dimensions 35.1A,67.4A,37.05 A, B=94°. They diffract to 1.5 resolution (Rsym=4.41%). Data from native and derivatives have been collected. MIR phasingis in progress. ii, Gastric lipases are pH resistant enzymes with a molecular weight of ca 50,000, including 15% sugars. Every glyco-variant from different species have been purified by ief, and submitted to crystallization, using parameters found by an incomplete factorial analysis method. All attempts were successful, but most crystals were unsuitable to Xray studies due to huge cell dimensions. Dog gastric lipase crystals, the most suitable for X-ray study (pH 6.8, 12% PEG 6000, 20°C; P2,2,2 :182A, 211A, 98 A), contain ca 8 moleculesin the asymetric unit , and therefore diffract to only 4A on lab sources. iii, Crystals of a complex between porcine pancreatic lipase (50000 Da) and its co-factor, colipase (11000 Da), have been obtained. These 1/1 complex crystals are disordered, and diffract weakly. Horse pancreatic lipase has been crystallized in space group P2,2,2,, (89A,97A,145A ; pH 6.0, PEG 8000 10%, 20°C)and a 2.3 A native data set has been collected. One PCMBS derivative was not sufficient to produce a good map, and weare still looking for other derivatives. Two crystal forms of human pancreaticlipase (purified from pancreas juice) have been obtained, different from the one reported in the litterature. They crystallize in the same conditions and space groups are P2, and Py. Native data sets have been collected.
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THE STRUCTURE OF HUMAN PANCREATIC LIPASE SUGGESTS A LOCALLY INVERTED, TRYPSIN-LIKE MECHANISMFurthercrystallographic refinement of the structure of human pancreatic lipase hasled to an improved model which has been used for modelling studies of the hydrolysis of triglyceride substrates. In addition to the removalof the flap, further changesin the protein structure around the active site appear necessary to formulate a stereochemically plausible mechanism. A locally inverted trypsin-like mechanism is presently favoured and demands relatively modest changes of the X-ray structure. Additional new findings include the interpretation of the difference density for a butylboronic acid derivative and the location of a Ca2+ bindingsite.
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THE CEC BRIDGE LIPASE PROJECTThe Commission of the European Community is implementing several priority actions specifically designed for improving the competitiveness of European Biotechnology. One of these actions aims at the establishment of a Community network for training and research and has been executed, since 1982, in the framework of three successive Community programmes BEP, BAP and BRIDGE. These activities are conducted via two different types of projects : N-projects and T-projects. The T-projects, larger targeted projects, are aiming at bottlenecks resulting from structural or scale. constraints. One of these, the T-project on "Characterisation of lipases for industrial applications : three-dimensional structure and catalytic mechanism"does go backto a successful transnational collaborative research effort on phospholipases in the Biotechnology Action Programme by groups in Marseille (F), Utrecht (NL) and Tübingen (FRG). The aim of the T-project should be to acquire so much new knowledge about a sufficient number of these enzymes that it will be possible to understand why they are lipases and how they function as such.
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Title_Foreword_Contents_List of authors_Photo of participantsLipases are a highly interesting class of enzymes, since they exhibit activity only at the waterlipid interphase. They are also of high practical relevance, since they are key enzymesin fat metabolism, and they can be utilized for various industrial applications such as detergents and chiral synthesis. Progress in both basic understanding and industrial use is hampered, however, by the lack in fundamental knowledge about lipase structure and function. As a result, the CEC has decided to support a 3 years’ program onthe structure analysis and protein engineering of lipases, in the framework of a BRIDGE program (see the following article of Dr. B. Nieuwenhuis). As a starting point, a CEC-GBF Workshop underlying this monograph was held in Braunschweig from Sept. 12-15, 1990, summarizing the present state of knowledge in this important area of biochemistry. Fortunately, researchers from most groups active in this field from around the world gathered at this workshop, as indicated in Fig. 1. As a result, the meeting permitted for an up-to-date assessment of the area, on a global scale. Since the contributions of all active participants were submitted in written form during the workshop, this monograph gives an excellent survey on the field of lipase structure, mechanism and cloning. At this point, I would like to acknowledge the invaluable contribution of several individuals who have greatly contributed to this endeavour. First, I would like to thank the two co-organizers and co-editors of this monograph, Prof. Lilia Alberghina of the Universita degli Studi di Milano and Dr. Robert Verger of the CNRS Marseille, for their most important contributions in the invitation of the specialists, the preparation and the organization of the workshop. Special thanks are due to the CEC, represented by the BRIDGE Lipase Project Leader, Dr. Benedict Nieuwenhuis, for both financial support and organizational help. The German government, through the GBF, its National Research Center for Biotechnology, has contributed strongly through financial subsidies and indirect help, mainly in organization. On a more personal note, I would like to acknowledge the assistance of Dr. Marianne Kordel in thefinal preparations of the workshop, of many students and assistants in our group, and in particular of Ms. Birgit Balster and Ms. Sylvia Lenk for taking care of the workshop secretariat and the preparation of this monograph. Finally, the experienced andskillful help of Dr. Johann Heinrich Walsdorff as the copy editor of this monograph is gratefully acknowledged.