Self-evolving microstructured systems upon enzymatic catalysis

Biochimie 11998) 80, 421435 © Soci6t6 fl'anqaise de biochhnie et biologic mol6culairc t Elsevier. Paris

Self-evo|v|ng microstructured systems upon enzymatic catalysis J Chopineaw ~, S Lesieur b, B Carion-Taravella ~, M Ollivon b a Laboratoire de Technoh~gie Enzymatique, CNRS-UPRESA 6022, 60206 Compi@ne: I~l:~luipe des Syst~mes Polyphasds. CNRS- URA 12 #8, 92296 Chdtenay-Mahtbo; France

(Received 17 November 1997; accepted 23 April 1998)

Summary -:-The consequences of cell microslructuration on enzyme functions is discussed in the framework of selbevolving microstmciured systems, Molecular assemblies of amphiphiles or Iipids are spontaneously fornled by seif-organisation. Among these dfft~l'eni structures, mver~ed inicelles, liquid crystalline nlesophases and vesicles are hosts lor enzymatic reaction studies, Inside a living cell, phospholipid metabolism is responsible for membrane structural modifications" the catalytic l~haviour o1' lipolytic enzymes, mainly phospholipase (PL) A2, is descfibeed in relation with structural aspects of biological membranes. The implication in cellular regulation events of PL C and PL D is discussed in relation with the role of their reaction preducts as second messengers in membrane fusion processes. The m vitn~ synthesis of dialkyl phosphatidylcholines, vki the enzymatic "salvage pathway' which leads to Ihe formation of vesicles upon phospholipid tbrmation, is considered in relation xvilh autopoiesis. Mote recent studies on self-evolving systems based on enzyme-surfactants reactions are detailed. The interactions between amphiphilic aggregates and enzymes allow to explore the OGt~-tanol/water phase diagram. Enzymatic formation of dipahnitoylphosphatidylcholine (DPPCI liposomes and non-ionic surfilctant vesicles (NSVi, starting from mixed micelles or open strtictures, finally sets an example of a biorrth~tleticself-evolving system. (© Soci6t6 l'ranqaise de biochilalie el biologic 111oldculairc/ Elsevier, Paris).

micelles t vesicles / membranes / enzymes / self.organisation / self-evolution Introduction In living systems most of reactions arc driven by enzymes. Indeed, enzymes are active tools of the cell fiictory in which they make most of the chenaical transformations II I. The restiiting products belong to two ca!egories: those of metabolic pathways needed for normal cell functioning and those responsible Ior cell structuration. A ln~0or part of the knowledge about enzymes resu!ls front the study of their

!uncuo in metabo!ie pathways, while their implication in structure building is poorly documented. Then, biotechnological impact of enzymes is directed towards production of chemicals through reactional schemes derived fi'om those of cell metabolism 121. Mass production of metabolic chemicals is achieved on industrial scale and enzymes in organic solvents are now well-recognised as catalysts by organic chemists I3-51. In contrast, self-assembling coin° pounds are only produced on a laboratory scale and also no example of biotechnological structure elaboration is known. Indeed, material scientists are trying to mimic the molecule self-assembly of living organisms which built all kinds of unbelievable sophisticated structures from the angstrom to the meter scale 161. Limiting ourselves to the cell dimension or less, we examine here what is known about enzyme activity, inside cells and their models, either implicated in or applied to the formation of supramolecular assemblies. Biological membranes are integral entities of the cells in which they delimit aqueous compartments having different compositions, the more refined ones being organelles. Thus,

they organise the living rnauer both by mamtanung ' ' " - differ° ent functions and metabolisms in each of the compamuents and by contro!!ing transport of various solutes between them. in this respect, many processes are t~dated to p!asmic membranes among which exocytosis, fusion, endocyiosis and intracellular trlilt'ic of bion/ttleculcs 171. Moreover, nienl-

l~ralles provide a iwoodinlensional matrix in which muliienz).o niiilh: i°eaclions such as ri.'.~pilahlry chain oi ~ pholosynlhctic l)roduction of ¢|!¢|gy take place l l, 8]. These asscmb!ics arc coinposcd mainly of lipids (40-50%) and proteins (50:60'%), the lipid/protein ratio depmldin 7 on the organelles. Their moDo ¢uhu° arl'angen!enl is well described by the "nletamorphic fluid nlosaic nlodel' 191. Since cells :ire really stuflTd with memo branes, in turn proteins, hldependeiltly of their riature, son!e of which are enzymes, lii'e submitted to compartmentalization ct'= fects and evolve in i~sti'icted domains oi ° spaces 110, I 11. This is evident for integral membrane proteins or membrane linked proteins, the motion of which is indeed bidhncnsionally i~ o stricted. Interracial proteins stick as iipases, phospholipases or G proteins and water-soluble proteins, the substrates of which are compartmentalised, are functioning in a three:dimensional organised matrix. Enzymatic functioning is usually studied on isolated enzymes in watel; Mechanisms of enzymatic catalysis elu: cidated from aqueous experiments are also generally ascer: tained by solid state crystallographic data established by X-ray diffraction 1121. Although this procedure presents inevitably major interest in enzymology, the environments of the corresponding experiments, whether isotropic and

422 highly diluted solutions or almost anhydrous media, remain far from in vivo microheterogeneous conditions. The consequences of cell microstructuration on enzyme functions and the need of geometrically restricted systems diversity of cell organih [ 13-161, The study of ~me basic physicochemical features as the structures found at the cel!ular level. These biomimetic microstructures are varying according to the native compartments (aqueous mr lipidic) fr,)m which enzymes are coming. Colloidal systems offer the pos.~ibility to have a welldefined chemical composition and precisely characterised structure. In the related studies, enzymatic activity was widely investigated by using static structured media while a cell is a highly dynamic entity. The next logical step was to develop systems, the structure of which can interact with enzymes. Indeed, ternary mixtures composed ot surfactant, water and organic solvent provide a variety of colloidal mrganisations capable of self-evolution through dynamic interactions of their components with an enzyme I 171. Such systems also allow to follow enzymatic reactions in restricted environments II81. Specifically, in this last case. enzyme is responsible for environment changes which in turn modulate enzyme catalytic activity. However, the chemical nature of the product(s) and the phase(s) formed are f~r from reproducing cell conditions. Indeed, the rationale of ~uch work would be greater if natural molecules and system.~ of bioiogical relevance were u,~d instead of organic solventwater based mesophases. In this respect, phospholipid(s).suro fa~tant(s) mixtures Ibrm numerous types of aggregates in water dependinl~ on their relative pn)l~)aions. Solubilising surt'a~tant (detergent) removal fi'om mixed micelles yields vesicle form~tion through the soocalled micelleovesicle transi~ don |19~241, Thi~ last i~ involv~ in many pt~)ces~es of lil~)o bion~mbrane p~0tein extraction and n this way, an enzymatic procedure for iiposom¢ I:)reparahon through micelle~vesicle transition wa~ recently proposed 1281. In this paper which focuses on en~ymology in microstructured media, we first consider the basis of molecular sdfoorganisation of amphiphilic compounds, Subsequent to int~ucing the major types of so-obtained assemblies, their applications to the study of en~ymmic reactions in welldefined and static mic~heterogeneous environments are surveyed, Then, the more recently develol~d self-evolving systems, based on dynamic interactions between amphiphilic aggregates and enzymes, are exhaustively described,

Mol~ulur selfoussembliesus potential biomimetic systems Micn~structured m~els tbr biological systems are possibly obtained taking advantage of amphiphilic or lipidic mole-

cule properties. Indeed, when mixed with water and/or organic solvent, these compounds spontaneously originate lyotropic phases in relation with the intrinsic ability of the molecules to organise themselves.

Amphiphile and lipid seif-organisation Most amphiphilic and lipidic molecules display a double affinity on one hand for aqueous (polar) and on other hand for organic media (non-polar). In water, an alternative way to phase separation is self-association which eliminates the energetically unfavourable contact between the non-polar part of these molecules and water, while retaining their polar part in an aqueous compartment. Generaily, ~mphiphile or lipid self-associatitm is strongly co-operative and produces molecular aggregates of very diftbrent sizes and geometries, mainly depending on various intrinsic parameters (hydta~philic/lipophilic balance, degree of ionisation, concentration, etc) and on external factors (temperature, pH, ionic strength, etc). Modification of aggregate morphology occurs either gradually within a single phase region or simultaneously with a phase transition. More precisely, the characteristics o1"the colloids formed by amphiphiles in aqueous or organic systems closely vary wi',h the nature and mutual interactions c~f the polar and non-polar moieties of the molecules. Parameters governing such assemblies are divided into three classes. In the first class are found parameters acting directly on polar groups and/or influencing their surroundings and interactions. The number and distribution of the polar units within the amphiphi!it molecule should be considered first as they govern accessibility to water and conformation of the amphiphile. Then, intrinsic ionic or non-ionic character and hydration degree, often depending on the water col|tent, appear as tile most important I'act~,'s as well as the itmic s!rength and pH of the continuous medium. In the second class are all the I~lctors affecting the packing of the non-polar backbones, ie for aliphatic chains, mai,ly their number, length and both level and position of their eventual insaturations. In the third class are parameters acting on the state of the system, more or less condensed, such as temperature and pressure.

Pat'kh~g paramewrs All parameters listed above govern the whole arrangement of the amphiphiles through complex interactions which are diMcult to sum up exactly. A relevant attempt to predict packing fl~)m only geometrical considerations was made several years ago 129-321, Providing the molecules considered are the juxtaposition of two covalently attached moieties of opi:~site polarities, the t y ~ of packing can be predicted in first approximation on the basis of the intenictions between the hydrophilic and hydrophobic groups of the molecules at each side of the water-oil interface. The critical packing parameter p = vial (v and I are the apolar group volume and thickness; a is the apolar group-water

423 interfacial vature of molecular ceiles are

area of llle molecLlle) reflects die radius of curthe water-oil inlcr|'ace which accoun|s for the aggregation s|ale (fig I~. For inslancc, dirccl miexpecled by molecules wilh large p~lar head

Amphiphiles

Packing parameter p=v/a|

groups (P < (}.5), smaltcr head ~, ,,rou p. or doubBc-chai~ m~le¢u[es {lypica~ly. 0.5 < P < I) self-assemble in ~esic[es. while even :,mailer pokir head groups ~P _>_ i} lead ~o the |ormation o f planar biiayers or invelted structures. How-

Shapo

Organ|sation

Phase

a

Single-chained amphiphiles with large polar head group

Micelles HexagonalI

p< 1/2 Cone

Single-chained amphiphiles with small polar head group or doubled-chained amphiphiles with large head group, fluid chains

Flexible l~e!!ar (vesicle)

l/2
Truncatedcone

Doubled-chained amphiphUes with small head group

p~l

Doubled.chained amphiphiles with small head group, nonionic amphiphiles, poly-unsatured chains

p>l

L~ellar Cubic Cylinder

i~nverted~truncate d

@

Reversed micelles HexagonalII

cone Fig I. Structures and phases of amphiphiles as predicled by their geometrical packing. The packing parameter p = v/al is expressed a~ a function of the hydrocarbon chain volume (v). the hydrocarbon chain thickness (!) and the hydrocarbon-water inlerfacial area (a) of the amphiphilic molecule. Adapted from lsraelachvili et a! 130-32l.

~4 ever. this theoretical approach d ~ s not easily allow the predlctl n of packing for mixtures of compounds having different vlal ratios. Only in the case of ideal mixing, a mean value of P was tentatively estimated from the different v/al

foreign molecules are too comi~iex to be taken into account. Besides. structures formed through energy supply and reaching out-of-equilibrium state, either reversible or not at the laboratory time scale, are not predictable from the conventional vial expression: for instance ultrasonic irradiation of planar multilayer assemblies of molecules characterised by a P value of i. yields small unilamellar vesicles stable over several months. Independently of this cons;deration, figure ! represents some of the basic structures that are tYequently tbund when these constituents are mixed in different proportions with aqueous soh,tions 1341. Indeed, the diversity of the shapes encountered represents an important reservoir tbr future enzymatic studies.

Molecular d(~¢sion An important feature of the lyotropic mesophases formed by amphiphiles is their fluid or dynamic nature which is not conside~d in the static geometrical description. Regarding biomembranes, this property responsible li~r motion and transport, i~ an important factor for enzymatic reactions 1.151, Namely, enzyme o~ration depends on the exchanges of materials, eg em,~ymaticsubstrates or produc~s, which can occur only thanks to molecular or supramolecular diffilsion processes, within (transversal or longitudinal diffusion) anger ~tween (transl,', coalescence) the compartments of the cells, The ~gulation of these pr(~esses is 0hen control° led by the natu~ and proportion of the membrane constituenis: ibr instance cholesterol modulates lipid dynamics in lamellar arrangements due to its condensing effect which reduces t~ghter n olecular packing and ~stricted motions of the hydr~arbon chains 136, 371. The dimensionality of the structu~s implicated is a governing factor as well, By hydrating one amphiphilic compound or several in adjusted mixtu~, it is possible to generate artificial edifices offering a more or less restricted f ~ d o m of molecular movements and thus capable of mimicking living media, While molecular motions at the interlace ~tween amphiphilic assembly and continuous phase are generally two-dimensional, the diffusion of one molecule within the core of the structu~ should de~nd on its degree of fi~eed~m, In this respect. it will ~ e x i t e d that micellar phases allow three-dimensional isotet)pic motions, lamellar or hexagonal packings impose two-dimensional displacements, while movements in cubic liquid crystals are extremely confined in all directions 1381, An im~rtant characteristic of such assemblies when formed from phospholipids is the possible existence

of phase transitions as a function of the temperature which mainly involves the passage of the hydrocarbon chains from a solid ordered to a liquid state. Thus, temperature variation allows to control the molecular diffusion rates. Moreover, assembly lifetime is an important parameter to consider when molecule building blocks are involved in reactions: for instance micelle lifetime is close to 10-3 s, vesicles c ~ reach several months, while the lifetime of multilamellar structures is almost infinite. Closed colloidal structures like vesicles, reverse or direct micelles [391, hinder interparticle exchanges of their internal solutes although the mobilities of both the solute inside the particles and particle itself could be very high. Thus, the rate of an enzymatic reaction, very fast in solution, will be slowed down by the selective encapsulation of either the enzyme or its substrate in vesicles and the pt~ocess kinetically governed by the transversal diffusion of the s~cies through the particle shell 140, 411, Microstructured systems used for enzymatic studies For a few decades organised microassemblies have been used to create microheterogeneous media for enzymatic studies. Enzymatic catalysis in colloidal systems has emerged from different preoccupations. Some of them locus on a chemical approach, the aim of which is the biocatalytic conversion of water-insoluble compounds such as triglycerides, fatty acid esters and steroids 1421. Others deal with the use of microstructured environments for studies of enzymes functioning, using lyotropic potentialities of amphiphiles in water and/or organic solvent mixtures 1431.

Reversed mi~'~,lh,s lnvestigalions of the structure o1' biomembranes evidence non°bilaycr lipid structures represented either by reversed mice!les in between the two monolayers of the bilayer or by inverted type I! hexagonal phase [44-46l. These structures are the basis of the metamorphic mosaic fluid membrane model and may correspond to the transitory molecular organisations initiating membrane disruption, for example during end~ytosis and exocytosis [441. Evidence for incorporation of different proteins (cytochrome c, methemoglobin and cytochrome P450) inside inverted micellar structures was obtained 147]. Enzymatic studies using hydrated reversed micelles of surl'actant (like those obtained with AOT/water/octane) were used for understanding the functioning of enzymes in natural lipid systems. Moreover, the use of reversed micellar systems for incorporation of water soluble proteins or interfacial enzymes is well documented (for reviews see 114-16]). The most important properties of reversed surfactant micelles arc the following: i) they are thermodynamically stable; it) they present an aqueous core separated by a surfactant barrier from the bulk apolar solvent; iii) their inner size (a few nanometers) is comparable to proteins' gyration

425 radius and can be modulated by the water conten|, and iv) their optical transpm~ncy make most specm~photometric investigations of proteins applicable. Once incorporated in reversed micelles, the guest molecules may display changes in conformation induced by electrostatic interactions of the protein with the polar head group of the surfactant molecules [151. Enzymes usually follow classical MichaelisMenten kinetics (the reaction rate is an hyperbolic function of the substrate concentration |!2]) but they may acquire characteristic alteration of their biological functions. Enzyme-structure reciprocal influence is also governed by the water content: catalytic parameters are dependent on the surfactant hydration degree ([waterI/Isurtiiclant]) which ix mainly responsible l'or the inner cavity size of the micelles. Reversed micelles based on phospholipid were used for enzymatic studies 148I, Structural inlbrmation concerning those micelles in relation with enzymes is scarce compared to the widely used AOT aggregates. However, in the latter case, the water pool is truly limited and its increase needs the addition of a cosurfactant which may complicate the interpretation of the enzymatic resu!t

Liquid co,stalline mesophases Enzymes Iphosphatases, peroxidasest can function in liquid crystalline mesophases based on surfactants, eg lamellar,

reverse hexagonal or cubic organisation i49I. The study of different enzymes in AO'Bwater-octane and Brij96-~.a~crcyclohexane systems Mtowed to cRassify them in t ~ u groups. The enzymes belonging to the first group present an anchor group on their surface ~carbohydrate or lipid moiety) which ensures the regulation of their activity by the nature of the surfactant packing. The interaction of enzyme anchor with the lipidic matrix may have the same role m vivo. The second group includes enzymes whose activity is not affected by the nature of the phase. For both classes the steady-state kinetics of enzymatic reaction obeys the Michaelis-Menten equation. The catalytic activity of soluble enzymes depends on the size of surfactant aggregates modulated by water content, even in the same phase. In particular. dramatic changes occur in reversed micelles, lmmobilisation of enzymes or nficroorganisms in such mesophases for bioconversion in organic solvents was ah'eady repolled I501. Lipid's polymorphism in water leads to structures vao rying from monolayers, lamellar phases to cubic phases as shown in figure 2, in which enzyme conformation and/or functioning were studied. Monolayers and bilayer.s arc mainly used for membrane protein reconstitution [511 while highly concentrated cubic phases were proposed for immobilisation of both membrane and water-soluble proteins. Indeed, hydrated cubic phases display ' ~teresting properties lbr studying proteins 152, 531. They ,re optically transpar-

..... ;..5;.......

e

d

]

'~.

Fig 2. Self-organisa!ion and supramolecular aggregates of amph iphiles in water: cyli ndrical mic¢llc (a); hexagonal i phase ( in finiu: cyli mlrical micelles) (b): cubic ! phase fi)rmed by closed direct micelles (e): bicontinttous cubic phase (prolamcllar l~hase) (d): hunellar pirate t i.l°initc bilayered sheets) (e); monolayer (13. Adapted |'toni 1981.

426 ent. They provide an organised matrix the basic structure of which is a molecular bilayer. Lysozyme and other globular proteins have been incorporated in monoolein-based cubic phases [541. Bacteriorhodopsin and melitin structural in lysophosphatidylcholine/water nation about the kinetics and mechanisms of the enzymatic reactions remains sc~ce. The principal drawback of the liquid crystalline phases is their high viscosity which jeopardises classic enzymology techniques [561. Vesicles

Vesicles are closed membrane capsules delimited by single or multiple lameilae that can be formed from a variety of amphipatic molecule..S Formation of vesicles can result either from spontaneous curvature of a lamellar structure or requires specific conditions (heating, mechanical constraints, solvent extraction, etc) which bring energy to the system. They are usually classified according to their size range, number of iamellae and available internal volume. One can discern muitilamellar vesicles or MLV (diameters from several hundred nanometers to a few microns), unilamellar vesicles, giant (GUV, diameters of several microns), large (LUV, diameters of several hundred nanometers) or small (SUV, diameters smaller than 50 nm). When constituted by lipid or phospholipid bilayers, vesicles are often called lie t~somes, The structure of liposomes offers the simplest tnt~del of two aqueous compartments separated by a membrane and a~ considered in this as the minimal cell model. The uses and applications of liposomes are versatile: they are used tbr membrane protein reconstitution as well as model systems in the study of biological recognition pro° ces.~es and of biomembrane dynamics 15%591. For applica~ lion purposes vesicles are simple aqueotls reservoirs or elementary volume units lbr the transport of water-soluble compounds including enzymes in their internal compart° merit while lipophilic molecules are conveyed in the bilayer. For practical applications of enzymesovesicle systems~ e.~ stabilisation of proteins under non-denaturing conditions, development of drug targeting systems or creation of microbio~actors, the lack of knowledge on specific enzyme. vesicle interactions deserves to be filled. Self-evolving systems via enzyme-phospholipid ~actlons Most of catalytic studies in microheterogeneous environment were performed using organised microassemblies of surfactant, water and al~lar solvent as host lor enzymes. Nevertheless, such media are not weli-recognised by biologists studying membrane-interacting enzymes, who prefer either natural membranes or phospholipid-based mesophases which would more adequately ~present in rive situation. Indeed, in rive molecular motion involved in

subcellular and cellular events results from individual phospholipid movements (transbilayer migration or flip-flop, rotation and lateral diffusion) as well as from overall membrane rearrangements (membrane fusion, exchange or disruption). For example, although proteins are synthesised with a signalling sequence coding for their final dt=;fination, their intracellular traffic and targeting involve dynamic formation and disruption of membranes [71. Phospholipid metabolism via enzymes is implicated ira many of the membrane events. Membranes are renewable material from tie nero synthesised phospholipid molecules by means of acyltransferases [601. Conversely, among other factors such as local pH changes, ionic strength modification and accumulation of membrane disrupting agents, products of enzymatic lysi, of phospholipids by phospholipases tire non-bilayer fo~ming lipids and act in membrane disruption processes. For instance, diacylglycerols 161,621 and lysophospholipids 163-651 behave as solubilising surfactants (detergents). Lipases and phospholipases are lipolytic enzymes hydrolysing 'long chain fatty acid esters' 1661. IApases are defined as *carboxyl-esterases acting on long-chain acyiglycerols' 167 I. Lipases are able to induce sequence of physicochemical events. For example, the simulation of physiological conditions of fat digestion by pancreatic lipases showed successive aggregation states from mixed micelles to cubic phase allowing fat absorption 1681. Phospholipases (PL) act on phospholipids. PLAI and PLA2 hydrolyse the carboxylic ester' bonds ot" the position I and 2 of the glycerol moiely, respectively, yielding !ysophospholipids. PLC and PLD hydrolyse p!msphoester bonds forming acylglyccrol or pllosphatidic acid, respectively (fig 3). In the !'t~llowing we focused on Ihc self evolving cn zy Ine~ph~sph~flipid bi olnJ|ll¢l ic sy s t e l l l s including phos~ pholipases and acyltransferases, ellzymes implicated in tile membrane metabolism, ic in the catab,nlhm aud in IIic bio* synthesis of phospholipids, respectively. Lqmse trod phospholOmse A2 interface interactions

Three°dimensional structure and catalytic activity of lipases were extensively documented (for reviews see [67, 69, 70]). Their activity is significantly increased at tile lipid-water interfilce, a phenomenon known as 'interlhcial activation'. The molecular basis of iipase catalytic function and activation at an oil/water interface attracted attention for applied and fundamental reasons. These enzymes handle waterinsoluble substrates which distinguish true lipolytic nroreins from esterases which act on soluble esters and which present no inteffacial activation. Lipases share this intcrfacial activation property with PLA2, however, with lwo cony pletely d i f t ~ n t molecular mechanisms as shown by X-ray crystallographic investigations 170, 711. Lipases undergo a conformational change in response to adsorption at the interface while no conformational change in the protein is observed fur PLA2. in this latter case, after a displacement

427

H~CO - C -R~

t o HOCH I o H2CO - P - O |

X

O" LysophosphoHpid

S~¢bs~rate/Product DPPC/pMm~toyl-lysoPC PC/~ysoPC Di|inoleoyl-PC/l-lino|eoyblysoPC D~o|eoy|-PC/l-oleoy|-lysoPC

x

H2CO - C -R t |1

k

! o ~,- C - OCtl " II o I

H2CO - C -R~

R2 - C]_OCH

I R, - C - O C H " it

O

H2co I P- o } x

It~COH

Phospholipa~eC ~

~)"

~ ~

o O

j

II H2CO - P * OH

!

Phosphol~paseD

O Phosphatidic acid

diacylglycerol Phospholipid

Substrate/Product PC/Diaeylglycerol

X: ethanolamine, choline, serine, glycerol or inositol

PE/Diaeylg!ycerol

Substrate/Product PC/Phosphalidic acid PE/Phosphalidic acid

DPPC/Diacylslycerol Fig 3, Ilyth'oly,.is ~,1' I~ht~.,Idlolilml carl~oxyli¢ c~.ler ~n' !~ht~,,IdlO~estcr link:l~,¢', calalv.,cd I~y l~ht.~pI.dil~a.,c~ A2, (' :tnd I ) ,,\n~v, ,, iml0c.t~' the hydrolysis sites located either in the plmspholii~id pohlr h e m gl'ottp (PLC and PI.I)) or al the water.oil interlace I¢v¢1 (P!.A2) It~l, ?r~, ,~3. 84, 99-11)4,I. of water molecules, phospholipid transfer occurs I'rom the substrate aggregate (micelles monolayers, vesicles or other biomimetic membranes) to the c~talytic site through a hydrophobic channel. For most iipases the active site is not accessible to the solvent; contbrmational modification of the proteins occurs when the hydrophobic lid displacement liberates the acces~ to the active site through the hydrophobic channel 167, 72-741. Information concerning catalyric events is known at the angstrom resolution level for several lipase,;. The reaction is dependen,, on the :~urface composition and on the packing of lipid molecules, properties which are qualified by the term 'quality o1" the interface'. However, these aspects and their relation with lipase activity still need more investigation. 1671. PLA2 is a large superlamily of distinct enzymes whose products are important for signal transduction processes. membrane remodelling and general lipid metabolism 1751.

Most sit,dies in term of structure and reb,ulaliol~ low~!rds

~esicular substrates concern an ubiquitou,~ 14okDa extr~ cellular PLA2 176-781 A MichaelisoMentcn model wa~ de° veloped to allow the study of preferential hydrolysis of certain phospholipids by PLA2 1771. The influence of membrane properties on PLA2 activity consists in an enhanced activity towards dipalmitoyl phosphatidylcholine (DPPC) dispersio, in the crystalline to liquid state transition region of the plloso pholipid chains 177 i. Lysophosphatidylcholil~c molar li°action from DPPC hydrolysis regulates enzyme activity, so that. z~sa function o1"the local producl concentration, a two tool perccmo age increase could result in a burst of the enzymatic yield, Indeed, PLA2 activity is modulated by membrane's composition which determines structure, curvature and dynamics (fluidity or packing constraints of the bilayer}. Dynamic product inhibition due to limited rate of lateral dit L fusion of the lipid in the bilayer is observed 1781.

428

Liposomejiasion induced by phospholipase C Fusion events are major morphological modifications occurring in cell dynamics and most involve transient rear[7, 79]. Fusion poly(ethylene glycol), freezing, drying, heating, ionic strength modification, biological macromolecules or virus, etc [80--821. Liposome fusion was widely studied. The difficulties in ob~rvation lead some authors to use GUV in order to monitor fusion by using light microscope I811. An interesting approach is the biochemical relevance of fusion related to phospholipid metabolism, its role in cell activation being clearly established. Secretory processes are known to be the result of diacylglycerol production from phospholipid cleavage by phospholipase C (PLC) in the membrane of neutrophils. Fusion of synthetic vesicles mediated by PLC was investigated as a model to show the relevance of diacyiglycerol (hydrophobic product of the enzymatic reaction) in this process 161.8~.851. In phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/choIcsterol (2:1:1 mole ratio) LUVs. diacylglycerol promotes the formation of bicontinuous cubic phase Q:,,4 [61,851. When the vesicles are treated with PLC in the presence of l0 mM Ca-',', two successive enzyme effects can be observed. The last one leading to vesicle-vesicle fusion is inhibited by the reaction product diacylglycerol. The, low one is related to bulk lipid hydrolysis, h~ situ production of diacylglycerol is required for the occurrence of the thsion protess; addition of diacyiglycerol to preformed vesicles is unable to produce filsion. Cl'vflesterol and PE presence concomit~mtly to PC in the hilayer, appears necessar~ for sig, nit'leant fusion occurring at low levels of pllospholipid hydroly,~i~ 183, ~41,

LO~osomeomedi~atcdsynthesis ~l'phosph.th!yl~'holim~ The reconstitution of an enzymatic process involving four enzymes leading to the synthesis of PC was performed u~ing proteoliposomes as enzymatic media 1861. The enzymes sn-glyc~:ml-3-phosphate acyltransferase, l-acylsn~glycerol-3ophusphate acyltransferase, phosphatidate phosphatase and cytidinediphospho-choline phosphocholine transferase i,,;olated fr~m rnicrosomes were incorI~rated into soy~an PC liposomes by the dialysis of mixed micelle method, The ratio tK2/proteins was five in weight ~hat corresponds to a molar excess of about 600 PC molecules per protein, The four-step synthesis of ~ starts from sn-glyceroi-3~phosphate, The synthesised ~ is mainly Io palmitoyl-2-oleoyl-!~hosphatidylcholh~e as driven by the corresponding substrates (paimitoyl and oleoyl coenzyme A), These substrates are water-soluble while the PC prodated is an amphiphilic molecule that will be incorporated into the liposome bilayers, As a consequence the total interface of the vesicles increases durit~g the reaction progress, A different effect was expected depending on the length of

the newly synthesised chain PC. When C6 chain incorporation is used, a resulting increase in the number of vesicles is deduced from light scattering experiments. This is predictable from theoretical consideration s concerning vial geometrical changes from long (C16, P = 1.1) to short (C6, P - 0 . 3 5 ) chains in a phospholipid molecule. This approach showedthe possibility to reconstitute multienzymatic pathway using liposomes, moreover, the newly synthesised PC molecules were responsible for an increase of liposome number. This rationale was transposed to GUVs containing the enzymes implicated in PC synthesis salvage pathway. This system is considered as a minimal cell model capable of sell-replication and so related to autopoiesis 1861.

Self-evolving systems via enzyme-surfactant reactions

TenuaO,phase diagram eaploration Octyl-[~-D-glucopyranoside (octyl glucoside, OG)/glucose in watertoctanol is a classical ternary system showing different domains and mesophases depending on the relative ratio between the components. When t3-D-glucosidase hydrolyses OG in glucose and octanol a modification of the chemical composition of the system occurs which may cause phase transitions II 7, 181.This property was used to explore the pha-,e diagram upon enzymatic hydrolysis which is a convenient method for a continuous exploration, The enzyme is able to modify its own micro-environment; in return the activity of the protein is modified by the phase evolutions. The isothernlal OG/glucose in water/octanol phase diagram was established at 30°C by discrete additions of ecru,. nol (fig 4), It exhibits two monophasic domaius: in tile waler rich corner Lt is a micellar solution of OG which ~preads from OG cmc (0,73 wt%) up to ahout 60 wt% OG; these concentrated micelles can incorporate 7 wt% of octanol. L,~ is a microemulsion containing a large proportion of octanol. The lamellar mesophase !~,~is a third domain containing around 10 wt% octanol and 55 wt% OG. in the X region a homogeneous phase domain is found which is not easily identifiable. Enzyme substrates COG, ~ater) and products (glucose, octanoi) are all together the components of the pseudo-ternary system, l~-D-glucosidase catalyses the stoechiometric reaction: OG ~-water --> glucose + {~tanol As the hydrolysis reaction proceeds the relative ratios of the different components are continuously changing. In most cases, the enzymatic activity was studied from onephase region to a biphasic system. The measurement of gk:cose production, by HPLC, leads to different activity recordings. Enzymatic hydrolysis of OG producing octanol and consuming water leads to unique pathways. Some of them are

429

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performed by dk~Ssis, dilution, a~td ge~ fitmttio~ chromao tography [57 [. An enzymatic procedure for liposome formation through mice[le-vesicle transition v~as recenttv. proposed ['~' ' _ S , 88, 89]. Addition of sur|actant-hydrotysing enzymes to lipidsurfactant water mixtures leads to closed bilaver structures whenever hydrolysis products do not interfere with the iL~rmation of a larnellar phase. A number of works have focused on the micelle-vesicle transition of PC-OG system [20, 21, 23, 90]. The general scheme for lipid dissolution from a lamellar phase to mixed lipid-surfactant micelles is a succession of aggregation s~ates in which the detergent binding to phospholipid bilayers is mediated by hydrophobic forces. Detergent partitions into the bilaver, until saturation level,- s are reached: at that time the bilayer begins to expel mixed micelles composed of lipid and surfactant (fig 5A). Vesicle l'ornlation takes place in three distinct stages, which are the symmetrical opposites of those ob, ct d dunng the dissolution process, However, in the process of vesicles formation, the size and the homogeneity of lipid vesicles obtained depends upon the rate of detergent elimination [23, 91 [. The principle of vesicle formation through enzymatic removal of the sttrfactanl is illustrated in figure 5B. S :~ " r e

Fig 4. Exploration of the octyl-~-D-glucopyranoside ~OGl/octanoltglucose-water ternary phase diagram using [~-D-glucosida,,,c [105!. Mixture compositions in the constituents are reported according to the Gibbs triangle representation (weight percent). White regions indicate two monophasic domains which are an OG micellar solution (L,), a microemulsion (L2) and a Lt~type liquidcrystalline lamellar phase. Dashed regions (X) show mainly polyphasic domains corresponding to non-determined structures. ~-D-glucosidase translbrms one molecule of OG and one of water in one molecule o1"octanol and one of glucose. One molecule of water being replaced by one molecule of gh,cose, the enzymatic pathways Irom any point of the phase diag,'am describe the lines symbolised by arrows (a to el. shown ill figure 4 by arrows, The kinetic patterns observed can be correlated to each phase transition. In order to define and to correlate precisely the kinetic events ;,llltl phase changes, we developed methods which continuously monio |or the reaction progress by differential scanning calorimetry, while media microstructure evolution was l'oliowed by recording turbidity at 40() nm 187]. Kinetic studies below and above OG critical micellar concentration have shown that the enzyme does not accept micelles as subs|rate but only the monomeric form of OG. The modifications in the enzymatic reaction rate which occur during the microenvironment transformations are a function of the different mierostructured phases present, but they are also dependent on local concentration and the availability of OG monomer.

Vesicle Jormation by enzymatic plvcesses Liposomes can be prepared by mechanical andA~rchemical procedures [571. Processes based on elimination of a solubilising surfactant (detergent) fi'om lipid-detergent mixed micelles are commonly used for substance encapsulation when mild conditions are required. Detergent removal from mixed micelles leading to liposome formation is generally

'

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DPPC-DG vesicle formation by amylogh,'osida.w reaclion on mixed micelles The first example was based on alkylglucoside DPPC micelle to vesicle transition knowledge. The study of DPPC (P = I) and DPPC/dodecyl-~-D~ghlcoside (DG. P = 0.6) vesicle solubilisation by dodecyl-~-D-malto side (DM. P = 0.4) was performed to establish the phase existence regions and to partially determine tile DPPC~DGDM phase diagram (fig 6A)[921, The lines indicate phase botlll~laries or local Mruclural changes. Each area con'esponds to changes in aggregate morphology andtor sy~t~m~ phase b e h a v i o u r depending on the DMtDPPC ~nd DGtDPPC molar ratios, Enzyme was chosen to remove the water-interacting sugar moiety of the detegent. Amylogluo cosidase hydrolyses DM. forming glucose. The presence of glucose does not interfere with the phase boundaries as in= dependently shown 1931. The arrow indicates the evolution pathway of an initial DM/DPPC = !.8 mixture as governed by the stoichiometry of the ,'eaction. Small angle X.ray scat= tering was performed on DPPC-DG-DM reconstituted mix° tures along the enzymatic pattern 1891. The presence of miceiles from i.8 to I. ! DMtDPPC molar ratio gives a scat° tering band while Bragg diffraction peaks indicate the evolution of lamellar sheets from i.! to 0 DM/DPPC ratio. These lamellar structures have a mean repeated distance of 60 A, in accordance with pure DPPC lamellar structures. Three main phase regions are of interest for this study (fig 6A). the 'pure micellar' above the solid line M, the domain below line L in which the presence of DPPC-DG-DM vesicles is ascertained, and the intermediate region between

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is ~rt+omled Ihmlks to its Irmlsformation by die e~+zymein p~.lu~ts compatible wilh a lipid Imnellar slructure, Idetl, detergent concentr~tion, M and L corresponding to the coexistence of micellar and lamellar structures, A ratio I D M / D P P C ! a g = 1,8 (at point C IDMIDPI~Iag m 1,5) was chosmt I'or starting all enzymatic es~riments, The same enzymatic mixture was added to dift~mnl DP~+DM mixed micelles for DPPC concentrations from 0,5 to 10 raM, and the effect of enzyme concentration on thc p ~ e s s was examined, A typical turbidity recording as a function of time is illustrated in figure 6B, The initial low turbidity level is characteristic o1" mixed micelles: the sha~ increase which follows is due to the formation of large agg~gates, then the turbidity stabilises at high OD values ~ t w ~ n 0,~ and 1,2, Quasbelastic light scaltering measuren~nls indicate the evolution of aggregates size from 80 to 600 nm in appa~nt mean diameter, compatible with vesicular assemblies,

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To demonstrate that vesicles were formed, the enzymatic procedure was ~rformed in presence of a water+soluble fluorescent probe calcein, The reaction mixturcs obtained at different steps of the piece,' ' s's were chromatographed on a gel exclusion column coupled with fluorescence on-line detection, Elution profiles at the final step (curve b ill fig 7A) attested the presence of two distinct peaks which correspond to free and entrapped calcein, Figure 7B shows a I'r~eze-fracture electron micrograph of DPI~ vesicles prepared by the enzymatic method, Unilamellar closed vesicles, mean size ranging from 20 to 80 ran, ale clearly visible and comparable to standard vesicles obtained by ultrasonication, These results demonstrate together the presence of aggregates alter enzymatic hydrolysis of detergent in DPPCDM mixed-micelles. These aggregates are DPPC-DG

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Fig 6. A. Partial pseudoternary DPPC-DG-DM phase diagram in excess water at 37°C 1921. The axcs correspond to the binary DPPC DM and DPPC-DG systems and excess water is implied and not represented. Solid lines L and M corresponding to effective phase limits and dotted lines related to secondary aggregation state tra,sitions were established from the drastic events characterising the turbidity variations recorded along DPPC+DG vesicles solubilization by DM. The arrow symbolises the enzymatic pathway when the initial state is a 1.8 DM/DPPC micellar solution in agreement with: i) the closeness of DG and DM solubility's in water at 37°C; iij the enzymatic hydrolysis of one DM molecule yields one DG molecule. Rectangles show the micellar (filled), lamellar (open) and coexistence domains checked by small-angle X-ray diffraction analyses. B. Smnple turbidity variation recorded at 37°C, upon DM hydrolysis by anayloglucosidase from DPPC+DM mixed micelles with initial surfactant to lipid molar ratio of i.8 (I DPPCI = 1.5 raM). The significant increase m the optical density (OD) depicts the passage fronl mixed micelles towards larger aggregates assimilated to vesicles 128, 891.

vesicles which can accommodate up to 1.8 DG/DPPC molar ratio as shown on recolistiluled mixtures by calorimetry and X+ray diffraction experiments 1891.

NSV formation through esterase reaction on polyoxyethyh, ne cholesterol derivatives In a second system the detergent was chosen to be trans° formed in cholesterol, a naturally occurring membrane component. Diglycerol hexadecylether (Cit,G2) is known for its ability to form bilayers and vesicles when associated with cholesterol while in absence of cholesterol it forms planar interdigitated structures 191,941. Poly(oxyethylene) cholesteryl sebacate diester (ChoI-POE) was used to solubilise Ct6G?.-dicetyl phosphate in bufl~r in order to get an isotropic clear solution of open micelle-like aggregates (5 mM total lipid concentration at 50°C, CIc,GdDCP/CholPOE:I/0.07/I%mol). Polyoxyethylene cholesteryl derivatives are known to solubilise Cit,G2 into open structures 194, 951. When an esterase solution is added to the surfactant solution, it hydrolyses chol-POE into cholesterol which is soluble in the lipids, polyoxyethylene and sebacic acid both soluble in water. The evolution of turbidity from low OD values to high values was observed to be similar as that ob-

tained with tile DPPC system. Enc~lpsulation of ca!cein was successfully perlbrmed. A negative staining electron micrograph of the aggregates alier reaction demonstrates struc° lures similar to vesicles obtained by ultrasonication [961,

Conclusion Biological membranes organise the living matter both by maintaining different functions and metabolisms in each of the compartments and by controlling transport of various solutes between them. Moreover, membranes provide a two-dimensional matrix in which multienzymatic reactions such as respiratory chain or photosynthetic production of energy take place. Their supramolecular arrangement is maintained by phospholipids. Phospholipid metabolism underlines mainly processes in which membrane modifications occur (fusion, endo- and exocytosis). The diversity of their molecular packing potentially offers numerous struc° tures for enzymatic studies, and even self-evolving micros t r u c t u r e s in r e l a t i o n with e n z y m a t i c reactions, Nevertheless, the difficulty remains in the following question: how to obtain microstructured biomimetic models tak+ ing advantage of the self-assembly property of amphiphilic

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p ~ n e e of I of non.entrapped calcein {elation ~ak~ !1)from ¢alcein encapsulated in the vesicles (elulion aks I) we~ ~rformed by USing tWO columns in series (TSK6000 and ~G4000 PW lye, Toyo Sotlak Elution profiles we~ ~ o r d e d on line by using a ci~ulating qeanz cell (Heilma) placed ht a s~ctrofluorimeter (flow rate, 1.0 mlJmin). Fluorescence emission ofcalcein was detected at 419 nm (excitation wavelength, 367 rim), Thine independent ~actions wel~ carried o,h t'lMIn |he same initial DP~-DM re{cellar solution (I.8 DM/DPPC molar ratio: IDPEI mS raM) and stopped at different stages of the eu~ymatte pn)cess, cor~spondin~ to DMtDPi~" (DGtDPPC) total molar ratios uf 0.33{ 1.47)(b), 1.04 {0,76) it) and 1,35 (I),45) {d); pu~ D P ~ vesicles prepaid by ultrasonic irradiation in tile p~sence of I mM calcein We~ used as standard 1891{a), B, Freezet'ractu~ ek.~tron micrograpil of DP~-DG vesicles obtained by DM hydndysis by alnyloglucosidase t'nml DPPC-DM mixed micelles, 7 mm= 200 rim,

~

molecules? From literature data, two workable strategies can be distinguished. The first possibility is the use of organised microassemblies of surfactant/water/apolar solvent mixtures which give direct, reversed micelles and lyotropic liquid crystalline mesophases having hexagonal {direct and reversed), iamell~ or cubic organ{sat{on. The second takes advantage of phospholipid polymorphism in water which, in adequate conditions, spontaneously forms hexagonal or lamellar phases and are usually used as models to understand biological membrane processes. It would be reasonable to extend the choice of the systems among other st~bmicronic structures like polymeric colloids and liquid crystals, emulsions or microemulsions. The microstructured systems divide into two categories: i) host systems acting as supports for bioconversion or enzyme function study; and it) sell-evolving systems, the structure of which is varying as a function of the enzymatic process. The second one approximates better to the biota{tactics, Fundamental approaches of modelling enzyme-membrane organ{sat{on have emerged from phospholipase mechanistic studies; the role played by diacylglycerois as products from phospholipases action was related to membrane fusion. Enzymology in microstructured media has given seminal results opening new prospects in relation to origin of live, vesicles or GUV being viewed as minimal cell models. Enzyme induced transformation of non-vesicle aggregates into liposomes represents an alternative method for encapsulation of labile substances but from a biochemical point of view it provides an insight into how enzymes may mediate structural trawls{lions in biomembranes. The diversity of enzymatic reactions can be used to build aggregates hut also to !~lodil'ythenl afterwards. Ill :all tl)c,'se"studies a complementarily betwee, physicochemical description of tile phases in presence and heterogeneous enzymology has proven to be n~cessary, Enzymolo~i~ts arc usually using aqueous media to study enzymes, even those involved in phospholipid metabolism and membrane physicochemists are busy understanding the role played by the different phospholipids and their relation with membrane proteins. Moreover, the control of the enzymatic reaction often requires to adjust the substrate as a function of the chosen pathway and specific compounds have to be synthesised. This implies the coopet~ltion of chemists. The principle of using self-organ{sat{on of amphiphilic molecules for tile creation of functional artificial microsysterns imitating biomembranes is not new. For 10 years at least, it has given rise to the interest of chemists. Membrane mtMels have been envisaged both as microreactors 1971 or to simulate biomembrane structure, dynamics and function catalysed by chemical reactions 1341. By considering the significant ~servoir offend by enzymes and their intrinsic biological role, the mimetic biochemistry appears as a worthwhile alternative to mimetic chemistry, lnterdisciplinarity between enzymology, biophysics, chemistry and physics should contribute to its further development.

433 Acknowledgments Thanks to F Gaille and JP Lcchah'c (('entre hucrunixcrsitairc dc Microscopic Electronique CNRS t!PR 9042 } fur micrographie~,.

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