Interaction of Phospholipase A2 from Naja melanoleuca Snake Venom with Monomeric Substrate Analogs

Transcription

1 bur. J Bochem. 132, (1983) ( FEBS 1983 Interacton of Phospholpase A2 from Naja melanoleuca Snake Venom wth Monomerc Substrate Analogs Actvaton of the Enzyme by Proten-Proten or Lpd-Proten Interactons? Jan H. van EIJK, Hubertus M. VERHEIJ, Ruud DIJKMAN, and Gerard H. de HAAS Laboratory of Bochemstry, State Unversty of Utrecht (Receved December 3, 1982) - EJB 6288 Unlke porcne pancreatc phospholpase A,, the enzyme from Naja melanoleuca does not dsplay bphasc knetc behavour at substrate concentratons around the crtcal ncelle concentraton. Ths snake venom enzyme was further nvestgated by drect bndng studes usng n-trdecylphosphocholne. Bndng of ths substrate analog to the enzyme was montored by usng equlbrum gel fltraton, equlbrum dalyss and ultravolet dfference spectroscopy. It s concluded that, n the presence of submcellar concentratons of n-trdecylphosphocholne, a lpd-proten complex s formed consstng of about 4 proten and 36 lpd molecules. + CaZ ons are requred for the formaton of ths complex. A model s proposed whch descrbes the formaton of ths type of complex. These lpdproten aggregates are held responsble for the non-hyperbolc knetc behavour of the snake venom enzyme towards monomerc substrates. Phospholpase A, s wdespread n Nature. The enzyme s abundant n pancreatc tssues as well as n the venoms of arthropods and snakes. Irrespectve of the source, the enzymes show a hgh degree of homology. An dentcal mechansm for the hydrolyss of monomerc phospholpds has been postulated [I]. The actvty of all phospholpases strongly depends on the physco-chemcal state of the substrate. The enzyme dsplays Mchaels-Menten knetcs when actng on monomerc substrates. In the presence of certan organzed lpdwater nterfaces, phospholpase A, shows actvtes whch can be three orders of magntude hgher than found for monomerc substrates. Ths results n a break n the velocty vs substrate concentraton plots at the crtcal mcelle concentraton of the substrate [2]. For the pancreatc enzymes t has been proposed by Verger et al. 131 that a partcular surface regon of the enzyme, called the nterface recognton ste (IRS), s responsble for the nteracton wth aggregated lpd-water nterfaces. A conformatonal change at the actve ste, accompanyng the bndng to aggregated substrates, s thought to be responsble for the ncrease of the enzymatc actvty upon passng the crtcal mcelle concentraton. For the snake venom phospholpases, dfferent explanatons for the hgh enzymatc actvtes n the presence of lpd-water nterfaces have been proposed (for a revew see Verhej et al. [4]). Many venom enzymes dmerze at hgh proten concentratons and t has been suggested that dmerzaton s functonal n enzymatc catalyss. By actve enzyme centrfugaton, Smth and Wells [5] showed that the Crotalus adamanteus enzyme acts n a dmerc state on monomerc substrates. For cobra venom phospholpase A,, Abbrevatons. HPTLC, hgh-performance thn-layer chromatography; Hepes, 4-(2-hydroxyethyl)-l -pperazneethanesulfonc acd; CMC, crtcal mcelle concentraton. Enzyme. Phospholpase AZ (EC ). whch s monomerc at catalytc concentratons, a substratenduced dmerzaton has been proposed [6]. For these reasons a comparatve study of hydrolyss of a short-chan substrate and a study of the lpd-bndng propertes seemed to be of nterest. We have chosen Nuju melanoleucu phospholpase A, n ths study because t can easly be purfed on a large scale allowng drect bndng studes. MATERIALS AND METHODS Phospholpase A, Phospholpase A, was purfed from Naja melanoleuca snake venom as descrbed by Joubert and van der Walt [7]. Fracton DE 111 was used n ths study. The enzyme exhbts a specfc actvty of 5500 U/mg n the egg-yolk assay (ude nfru). Bovne pancreatc prophospholpase A, was purfed as descrbed by Dutlh et al. [8]. The covalent bovne prophospholpase A2 dmer, wch s a by-product of the tetrantromethane modfcaton of ths proten, was prepared as descrbed by Meyer et al. [9]. The porcne pancreatc phospholpase A, was purfed as descrbed by Neuwenhuzen et al. [lo]. Phospholpase Assay The enzymatc actvty was routnely measured n an eggyolk assay by contnuous ttraton of lberated fatty acds, essentally as descrbed by Neuwenhuzen et al. [lo]. The ncubaton mxture contaned one egg yolk n 300 ml H20, 6 mm CaC1, and 1.67 % Trton X-100. The actvty of the N. melanoleuca phospholpase A, on dheptanoylglycerophosphocholne as substrate was determned ttrmetrcally at ph 8.0 and 25 C. The ncubaton mxture contaned CaCl, (50 mm), NaCl (100 mm) and Trs

2 184 (0.5 mm) and varable substrate concentraton. The actvty of the porcne pancreatc phospholpase A2 on dheptanoylglycerophosphocholne was determned ttrmetrcally at ph 7.0 and 40 "C n the presence of 0.5 mm Trs, 100 mm NaCl and 1 mm CaC12. Double-dstlled water was used for the assay buffer and all glassware was cleaned n bchromatesulfurc acd. In order to check the buffers for the presence of surfaceactve contamnatons, surface tenson measurements were performed usng the Wlhelmy plate method [Ill. Determnaton of the Crtcal Mcelle Concentraton The crtcal mcelle concentraton (CMC) of the lpds, dssolved n the buffer used for the experments, was determned at 25 C by the Wlhelmy plate method [ll]. The followng values were determned : n-trdecylphosphocholne (0.28 mm) and dheptanoylglycerophosphocholne (1.6 mm). Proten Concentraton Proten concentratons were calculated from the absorbance at 280 nm usng a value of 23 for A&. Alternatvely the proten concentraton was calculated from the enzymatc actvty. Purfcaton and Radoactve Labelng of' n- Trdecylphosphocholne n-trdecylphosphocholne, obtaned from Medmarck (FRG), was purfed by column chromatography on slca gel usng a lnear chloroform/methanol gradent as eluent. The product was pure as judged from HPTLC usng chloroform/methanol/water (65 : 35 : 8 by vol.) as solvent system. Radoactve l4c-labe1ed n-trdecylphosphocholne was prepared by the demethylaton-methylaton procedure descrbed by Kamp et al. [12]. The specfc radoactvty of the fnal product was 2.35 x lo1' ds. mn-' mol-'. Equlbrum Dalyss Equlbrum dalyss experments were performed at 25 "C usng a Danorm dalyss apparatus (Dachema, Zurch, Swtzerland), A dalyss cell conssts of two compartments of 0.25 ml, separated by a sempermeable dalyss membrane (MI cut off > 5000). To one compartment was added 200 pl of proten soluton (10-20 pm of fracton DE 111) and to the other 200 p1 of lpd soluton (14C-labeled n-trdecylphosphocholne). The buffer used contaned 50 mm Hepes ph 7.5, 100 mm NaCl and ether 25 mm CaCl, or 1 mm EDTA. After equlbrum was obtaned (5-7 h, 100-pl samples from each compartment were added to 5 ml of a lqud scntllaton soluton (Packard Instagel). Radoactvty was determned n a Packard lqud scntllaton system. Equlbrum Gel Fltraton Equlbrum gel fltraton was performed as follows. A column (0,9x30 cm) of Sephadex G-100 superfne was equlbrated wth buffer (50 mm Hepes ph 7.5,lOO mm NaCl and ether 25 mm CaCl, or 1 mm EDTA). In order to estmate the molecular weght of the lpd-proten complex, the column was equlbrated wth buffers contanng a varable concentraton of n-trdecylphosphocholne. After equlbrum was obtaned, a mxture of N. melanoleuca phospholpase A, (0.2 mg of fracton DE 111), bovne prophospholpase A, (0.75 mg) and the covalent prophospholpase A, dmer (0.75 mg) was passed through the column. Snce the eluton volume of the prophospholpase A, and the covalent prophospholpase A, dmer s not nfluenced by the presence of monomerc or mcellar substrate analogs, these protens can be used as molecular weght markers [13]. The molecular weght of the lpd-proten complex was calculated from the eluton volume, usng a molecular weght of and for the prophospholpase A, and the prophospholpase A2 dmer, respectvely. Occasonally the lpd/proten rato of the complex formed on the column was determned. Therefore the concentraton of n-trdecylphosphocholne n the eluton buffer was rased to 1 mm, a concentraton hgh enough to saturate the proten wth ths lpd. 2 mg proten dssolved n 200 p1 column buffer were passed through the column and the phospholpase actvty and the lpd concentraton n the column fractons were determned. From the eluton volume of the lpd-proten complex, the molecular weght was estmated and from the excess lpd n the proten-contanng fracton the lpd/proten rato was calculated. Ultravolet Dfference Spectroscopy Bndng of n-trdecylphosphocholne to the enzyme was measured by ultravolet dfference spectroscopy, usng an Amnco DW-2A double-beam spectrophotometer, equpped wth a MIDAN TM mcroprocessor, nterfaced to Apple I1 for data handlng and storage [14]. The proten soluton (11.7 pm DE I11 n 50 mm Trs/HCl buffer ph 7.5, 100 mm NaCl and 10 mm CaCl,) was ttrated wth n-trdecylphosphocholne. In order to obtan free lpd concentratons, the total lpd added to the proten was corrected for the amount of lpd bound to the proten by usng the followng expresson : where LFREE =free lpd concentraton, LToTAL =total lpd concentraton, A293 = lpd-nduced dfference sgnal at 293 nm, AE: = lpd-nduced dfference sgnal at 293 nm at saturatng lpd concentratons, n = lpd/proten rato of the lpd-proten complex determned by equlbrum gel fltraton and eo =proten concentraton. RESULTS The hydrolyss of dheptanoylglycerophosphocholne by the porcne pancreatc enzyme s shown n Fg. IA. Whereas below the crtcal mcelle concentraton (CMC) a normal Mchaels-Menten curve s obtaned, a well defned jump n the actvty s observed upon passng the CMC. Ths result s clearly dfferent from the one obtaned for the Naja melanoleuca enzyme (Fg. 1B). Below the CMC the snake venom enzyme, whch s about 100 tmes more actve than the porcne enzyme, shows hgh actvtes, ncreasng almost lnearly wth substrate concentraton. No sudden ncrease n actvty s observed near the crtcal mcelle concentraton but the enzyme reaches ts hghest actvty about there. The actvty curve s almost rectangular and devates from a normal Mchaels- Menten hyperbola. Ths behavour mght ndcate that the N. melanoleuca enzyme does not form a smple 1 : 1 complex but aggregates nto a hgh-molecular-weght lpd-proten complex n the presence of monomerc substrates. In order to check ths

3 185 I I I I I I I I A LO I3 I I I I,/L--*-*-*- /* J ' CMC L IS1 ImMI IS1 (mm1 Fg. 1. Hydrolyss of dheptanoyl glycerophosphocholne catalyzed by (A) porcne pancreatc or (B) N. melanoleuca phospholpase A,. Expermental condtons: (A) ph 7.0, temp. 40 C, 1 mm CaCI,, 0.5 mm Trs, 100 mm NaCI, 1 pg proten/ml; (B) ph 8.0, temp. 25 "C, 50 mm CaCI,, 0.5 mm Trs, 100 mm NaC1, 10 ng proten/ml n-trdecylphosphocholnel lmm1 Fg. 2. Auerage number of lpd molecules bound 10 lhe N. melanoleuca phospholpase A, determned by equlbrum dalyss as a functon ofthe n- trdecylphosphocholne concentraton. The buffer contaned 50 mm Hepes ph 7.5, 100 mm NaCl and ether 25 mm CaCI, (0) or 1 mm EDTA ( x ). The proten concentratons were 18.7 pm (0) and 16.5 pm ( x) respectvely 2 7 4Q 0 / / CMC +d-x - I I I I I n-trdecylphosphocholnel (pm1 Fg. 3. Estmaton of rhe molecular weght of rhe lpd-proten complexes formed at 25 "C on a Sephadex G-100 suprrfnr gel fltraton column as (I ftncton of the n-trdecylphosphocholne concentraton. The eluton buffer contaned 50mM Hepes ph 7.5, 100mM NaC1, 0-300pM n-trdecylphosphocholne and ether 25 mm CaC1, (0) or 1 mm EDTA ( x ) possblty the nteracton of the enzyme wth a monomerc substrate analog was studed by equlbrum dalyss and gel fltraton. Equlbrum Dalyss The results of the equlbrum dalyss experments (Fg. 2) show that n a buffer wthout Caz+ ons the proten bnds only one molecule of n-trdecylphosphocholne. At hgh lpd concentratons near the crtcal mcelle concentraton the average number of bound lpd molecules ncreases sharply. In a Ca2+-contanng buffer the average number of lpd molecules bound/proten molecule ncreases sgmodally (Fg. 2) to a value of about 9 at the crtcal mcelle concentraton of n-trdecylphosphocholne (0.28 mm). The S- shaped bndng curve suggests that the complex formaton n the presence of Ca2+ ons s a cooperatve process (Hll coeffcent 2-3). The complexes formed by the N. melanoleuca enzyme were further characterzed by equlbrum gel fltraton. Equlbrum Gel Fltraton In order to analyze further the lpd-proten complexes formed n the presence of monomerc concentratons of n- trdecylphosphocholne, the sze of the complexes was estmated by gel fltraton. In the absence of lpd the enzyme s monomerc. Proten aggregaton could not be detected ether n the presence or absence of CaZ+ ons at proten concentratons up to about 0.3 mg/ml. In the presence of Ca2+ ons a sgmodal ncrease of the molecular weght s observed when the n-trdecylphosphocholne concentraton n the eluton buffer s rased to the crtcal mcelle concentraton (Fg. 3). The ncrease n the average molecular weght from to parallels the ncrease n the average number of lpd molecules bound to the proten as determned by equlbrum dalyss (Fg. 2). Ths suggests that both observed processes are the result of the formaton of lpd-proten complexes. From the molecular weght and the lpd/proten rato at the crtcal mcelle concentraton t was calculated that the fnal complex contans about 4 proten and 36 lpd molecules. In the absence of Ca2+ ons the apparent molecular weght remans approxmately constant and ncreases only at the crtcal mcelle concentraton, when a complex wth mcellar lpds s formed (Fg. 3). The molecular weght of ths complex does not dffer sgnfcantly from the molecular weght observed n the presence of CaZ+ ons. However, the stochometry of the complex formed n the absence of Caz+ ons could not be determned by equlbrum dalyss, because mcelles of n- trdecylphosphocholne do not pass a sem-permeable membrane. Therefore equlbrum gel fltraton was used to determne the lpd/proten rato of the complexes formed at saturatng concentratons (1 mm) of n-trdecylphosphocho-

4 186 Table 1. Characterzaton qf the lpdproten complexes formed wth n-trdeeylphosphocholne Parameter Molecular weght" Lpd/proten rato 9b.c 43' Calculated composton : proten molecules 4 2 lpd molecules molecular weght n-trdecylphosphocholne 150 pm 250 pm concentraton at (=)cmb)~,~ (z CMC)" half saturaton Hll coeffcent 2.6d p2.6" Value obtaned from Fg. 3 Value obtaned from Fg. 2 ' Value obtaned by equlbraton gel fltraton Value obtaned from Fg In Trdecylphosphocholnel (pm) Fg. 4. Lpd-nduced ultravolet djjerence sgnals ut 293 nm of the N. melanoleucaphospholpase A, asa juncton ofthe total (A) or thefree (B) n- Erdecylphosphocholne concentraton. The buffer contaned 50 mm Hepes ph 7.5, 100 mm NaCI, 10mM CaCl,, 11.7 pm phospholpase A, and pm n-trdecylphosphocholne. Y= fracton of total number of bndng stes whch are flled lne. The complexes formed were analyzed for proten and lpd content and ther molecular weght was estmated. Table 1 summarzes the propertes of the lpd-proten complexes formed n the presence and absence of Ca2 + ons. In a buffer wthout CaZ+ ons a lpd/proten rato of 43 was determned; from ths rato and from the molecular weght (about 70000) t was calculated that ths complex contans 2 proten molecules and 86 lpd molecules. In the presence of Caz+ ons a lpd/proten rato of only 9 was determned. From the molecular weght of the complex (agan about 70000) and ths rato, t was calculated that the complex s composed of 4 proten molecules and 36 lpd molecules. Ths result confrms the lpd/proten rato determned by equlbrum dalyss and ndcates that, n the presence of Ca2+ ons, complex formaton s almost completed at the crtcal mcelle concentraton. Ultravolet Dj'erence Spectroscopy When submcellar concentratons of n-trdecylphosphocholne are added to a soluton of the N. melanoleuca phospholpase A,, complex formaton can be followed by ultravolet dfference spectroscopy. The lpd-nduced absorpton band at 293 nm can be used as a senstve method to montor qualtatvely the nteracton between phospholpase A, and lpd n the presence of Ca2+ ons [13]. In Fg. 4A the ultravolet dfference sgnal at 293 nm s plotted aganst the total (free +bound) lpd concentraton. Comparng ths curve and the sgmodal curve of Fg. 2, t appears that the shapes are smlar but that the maxmal ultravolet dfference sgnal s reached at a hgher lpd concentraton. It must be realzed, however, that n equlbrum dalyss experments (Fg. 2) the free lpd concentraton s plotted, whereas n Fg. 4A the total lpd concentraton s plotted. Therefore the total lpd concentratons were corrected for the amount oflpd bound to the proten as descrbed under Materals and Methods. After ths correcton, usng a lpd/proten rato of 9, the curve n Fg. 4B was obtaned. Ths curve s smla. to the one obtaned by equlbrum gel fltraton and equlbrum dalyss, ndcatng agan that n the presence of Ca2+ ons a lpd-proten complex s formed below the crtcal mcelle concentraton. When the lpd bndng data were plotted n a Hll plot, a Hll coeffcent of 2.6 was calculated for the cooperatve formaton of ths complex (Fg. 4, nset). DISCUSSION As shown n Fg. IA, two dstnct regons are observed n the actvty profle of porcne pancreatc phospholpase A, actng on dheptanoylglycerophosphocholne. Below the crtcal mcelle concentraton, the enzyme forms a 1 : 1 complex wth the substrate and shows normal Mchaels-Menten knetcs. Upon passng the crtcal mcelle concentraton, a jump n the enzymatc actvty s observed when a lpd-proten complex s formed [2]. Bndng studes wth mcellar n- alkylphosphocholnes have ndcated that complexes are formed usually contanng two proten molecules and about half of the lpd of the orgnal lpd mcelle [ The actvty profle for the Naja melanoleuca enzyme actng on dheptanoylglycerophosphocholne (Fg. 1 B) s not bphasc. The hgh enzymatc actvty mght be due to a hgh turnover number of the venom enzyme towards monomerc substrates. However, the absence of a break n the enzymatc actvty curve at the CMC could also be explaned f the N. melanoleuca enzyme already forms a hgh-molecular-weght lpd-proten complex below the CMC. Drect bndng studes descrbed n ths paper usng submcellar concentratons of a substrate analog demonstrate ndeed that the venom enzyme does not form a smple 1 : 1 adduct. Instead, n the presence of monomerc concentratons of n-trdecylphosphocholne and + CaZ ons, lpd-proten complexes are formed contanng on average 4 proten molecules and 36 lpd molecules. We realze that the expermental condtons n the drect bndng studes are not dentcal to those of the knetc experments. Especally wth catalytcally very actve enzymes, such as the N. melanoleuca phospholpase, the amounts of enzyme requred n knetc studes are much lower than those needed n drect bndng expertments. In spte of ths lmtaton, t s temptng to explan the hgh enzymatc actvty of the venom enzyme at submcellar substrate concentratons by the formaton of smlar lpd-proten aggregates as observed n the drect bndng studes : premcellar lpd aggregates stablzed by several proten molecules.

5 187 Consderng the small sze of the proten, the bndng of 9 lpd molecules at dfferent ndependent bndng stes on one proten molecule seems hghly mprobable. Moreover, the cooperatvty of the bndng curves, together wth the knetcally observed very hgh enzymatc actvty at submcellar concentratons, suggest that the enzyme nduces lpd aggregaton. Therefore we propose the followng mechansm for the formaton of the lpd-enzyme complex. Although the N. melanoleuca phospholpase A2 shows some tendency to aggregate at hgh proten concentratons [7], the enzyme s a monomerc proten at the concentratons used n knetc and drect bndng experments. Addton of monomerc lpd solutons results n a cooperatve bndng of lpd molecules to the hydrophobc surface regon of the proten, thereby ncreasng the apolar surface area. The tendency to aggregate s ncreased and the proten molecules form a lpd-proten complex. Drect bndng studes usng n-alkylphosphocholnes have been carred out for both snake venom [17,18] and mammalan pancreatc [14-16,191 phospholpases. Araujo et al. [15] studed the nteracton between the porcne pancreatc phospholpase A, and mcellar solutons of n-hexadecylphosphocholne. The authors concluded that hgh-molecularweght lpd-proten complexes are formed by nserton of two enzyme molecules nto a lpd mcelle followed by an expulson of excess lpd molecules. In ths paper we have shown that phospholpase A2 from N. melanoleuca nteracts wth monomolecular dspersed n-trdecylphosphocholne molecules, resultng n the formaton of a hgh-molecular-weght lpdproten complex. Assumng that no premcellar aggregates occur below the CMC of the substrate analog, these lpdproten complex must be formed along a comcellzaton pathway. Drect bndng studes wth monomerc concentratons of n-alkylphosphocholnes and both pt vper [IS] and cobra venom [I71 phospholpases have been carred out, The change n the crcular dchroc spectra n the aromatc and farultravolet regons were used to determne bndng. Snce spectral changes were observed manly at detergent concentratons below the CMC, the authors concluded that they were studyng bndng of monomers to the enzyme. Snce the stochometry of lpd and proten n the resultng complexes was not determned, t mght very well be that the authors observed aggregaton rather than monomer bndng. Several authors have suggested that, apart from a functon n enzymatc catalyss, Ca2+ ons also play a role n the bndng of phospholpases to aggregated lpds [3,6,16, In the absence of Ca2+ ons, the lpd-bndng propertes of the N. melunoleucu enzyme resemble those of porcne pancreatc phospholpase: below the CMC both enzymes bnd only one lpd molecule, whereas upon passng the crtcal mcelle concentraton a hgh-molecular-weght lpd-proten complex s formed contanng 2 proten molecules and about lpd molecules (see [I61 and Table 1). The presence of Ca2+ ons s absolutely requred for the N. rnelanoleuca phospholpase A, to form a hgh-molecular-weght lpd-proten complex wth monomerc lpds. Ths latter result ndcates an ordered mechansm wth Ca2+ bndng frst before the enzyme aggregates nto a hghly actve lpd-proten complex. An ordered mechansm for the Crotalus adamanteus phospholpase A2 actng on monomert dbutyrylphosphocholne has been reported by Wells [22]. Although for ths enzyme actng on monomerc dbutyrylphosphocholne a hyperbolc actvty profle has been reported at low substrate concentratons, a further sgmodal ncrease of the enzymatc actvty below the CMC ndcates that ths enzyme also becomes already actvated at submcellar substrate concentratons. Although pancreatc phospholpases are able to hydrolyze monomolecularly dspersed lecthns by formng a 1 : 1 enzyme-substrate complex, the enzymatc actvty s only very low as compared to the rates obtaned wth mcellar substrates when hgh-molecular-weght lpd-proten complexes are formed. Snce the actvty profle for the snake venom enzyme, actng on monomerc dheptanoylglycerophosphocholne, devates from a normal Mchaels-Menten hyperbola, t seems unlkely that the hgh enzymatc actvty orgnates from a smple 1 : 1 enzyme-substrate complex. Therefore, we assume that also n knetc experments, usng much lower proten concentratons than for drect bndng studes, smlar hghmolecular-weght lpd-proten aggregates are formed n the presence of monomerc substrate concentratons. Snce both lpd and proten aggregaton occurs upon complex formaton, one can ask the queston: s the hgh enzymatc actvty observed n these aggregates caused by proten-proten or by lpd-proten nteracton? Several snake venom enzymes have been reported to be actve only n a dmerc form [5,6,23]. A proten aggregaton nduced by substrate mcelles has been reported by Denns et al. [24] for the otherwse monomerc Nuju nuju naja phospholpase A2. Consderng the stochometry of the lpd-proten complex of N. melanoleuca phospholpase A2 and n-trdecylphosphocholne, t seems nevtable that proten-proten contacts do occur. Such protenproten nteractons could nduce the proten conformaton responsble for the hgh enzymatc actvty of the complex. In many olgomerc enzymes proten-proten nteractons are mportant for enzymatc actvty, especally n allosterc enzymes where proten-proten nteractons result n cooperatve substrate bndng. On the other hand, the number of 36 lpd molecules mght very well be bg enough to form a core thus creatng a lpd-water nterface. In ths nterface a preferred conformaton of the aggregated substrate molecule could lead to hgh reactvtes as suggested before (25,261. Moreover nteracton of proten molecules wth ths lpdwater nterface mght nduce changes n the proten conformaton, thus mprovng the archtecture of the actve ste [31. These nvestgatons were carred out under the auspces of The Netherlands Foundaton for Chemcal Research (S.O.N.) wth fnancal ad from The Netherlands Organzaton for the Advancement of Pure Research (Z.W.O.). Ths work was supported by NATO grant REFERENCES 1. Verhej, H. M., Volwerk, J. J., Jansen, E. H. J. M., Pujk, W. C., Djkstra, B. W., Drenth, J. & de Haas, G. H. 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