Biochimica et Biophysica Acta. 1126(1992) 95-104 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2760/92/$05.0(~

95

BBALIP 53921

Competitive inhibition of lipolytic enzymes VIII: inhibitor-induced aggregation of porcine pancreatic phospholipase A2 A . M . T h . J . D e v e e r ", A . T . d e n O u d e n b, M. V i n c e n t c, j . G a l l a y ¢, R. V e r g e r d, M . R . E g m o n d '~, H . M . V e r h e i j ~ a n d G . H . d e H a a s e Unilccer Research Laboratory, Vlaardingo~ (Ncff,~erlan:ls), ~ Department of Physical and Colloid Chenusrry, Debije lint/tare, State Unirersity of Utrecht, Utrecht (Netherlands), c Laboratoire pour I'Utilisation du Ra),onnemen: Electromagnetique.CNRS, Orsay (France/, d C.N.R.S., C.B.M.5., Marseilh' (France) and "Department of Enzymology a'~d Protein Engineering, CBLE, State Unirersity of Utrecht, Utrecht ~Netherlands) (Received I0 January 1~2)

Key words: PancreaticphosphotipaseA,: Competitive inhibition; 2-Acylaminophospholipid; Light scattering;Time resolved fluorescence spectroscopy

Several 2-acylaminophospholipid analogues have been demonstrated to behave as potent competitive inhibitors of porcine pancreatic phospholipase A: (De Haas, G.H., Dijkman, R., Ransac, S. and Verger, R. (1990) Biochim. Biophys. Acta 1046, 249-257). Their inhibitory power appeared to be strictly controlled by the stereoconfiguration around the chiral C-2 atom and effective inhibition of the enzyme was ol~servcd only when incorporated into a miee!lar substrate-water interface. In the present study various direct binding techniques were applied to investigate the interaction of the enzyme with pure micelles of the stereoisomeric forms of 2-tetradeeyl-amino-hexanol-l-phosphocholine (R-C~-PN and S-C i4-PN). Upon equilibrium gel filtration of the enzyme (monomeric molecular mass--14 kDa) on calibrated Superdex columns running in m/cellar solutions of R-Ct4-PN, the phospholipase eluted as a lipid-protein complex of 74 kDa, Under identical conditions, m/cellar solutions of S-C,~-PN did not give rise to high-molecular mass aggregates and the enzyme eluted at its normal 14 kDa position, Light scattering cxperiments, ultrasedimentation and time resolved fluorescence spectroscopy studies confirmed the formation of a high-molecular mass aggregate between en~'me and R-CI4-PNmicelles. The ultimate complex was shown to consist of four protein and about ten inhibiter molecules. Using time-resolved fluorescence ~7c~u'o~opythe interaction was studied between the active site of phospholipase A2 and R-Ct4-PN molecules, both incorporated in an inert lipid matrix.

Introduction

in the original model of Verger et al. [1], describing the action of a lipolytic enzyme on a lipid-water interface, two successive steps were assumed. Binding of the enzyme to the interface is followed by binding of a single lipid molecule into the active site of the enzyme. According to this model it can be expected that the presence of a strong competitive inhibitor in the substrate-water interface not only will decrease the catalytic breakdown, but can also affect the equilibrium of the first binding step. In a recent series of papers [2-7]

Abbreviations: PLA 2, phospholipase A2; CMC, critical micelle concentration. Correspondence and present address: A.M.ThJ. Deveer, Unilever Research Laboratory, P.O. Box 114, 3130 AC, Vlaardingen. Netherlands.

we demonstrated that 2-acylamino-2-deoxy lecithin analogues are potent competitive inhibitors of phospholipase A 2 (PLA 2) and a kinetic model was developed to evaluate the inhibitory power (Z) of these phospholipid analogues. Incorporated into an organized lipid-water interface 1-alkyl-2(R)-acylamino phospholipids were found ~o be the mos~ effective competitive inhibitors, The stereoisomeric 2-(S)-anoIogues hardly possess inhibitory properties. The interaction of PLA 2 with miceiles of these enantiomeric inhibitors was studied recently by ultraviolet difference spectroscopy [8]. The dissociation constant of the interfacial PLA2-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than for micelles of the (S)-antipode. A lipid to protein stoichiometry of 2 was found for the complex of PLA 2 with the (R)-isomer. PLA 2 in which the active site residue His-48 is covalently modified by 1-bromo-2-octanone, also binds to the inhibitor micelles and in this

96 case identical dissociation constants were found for the stereoisomeric forms of the inhibitor. The main difference between octanon-PLA z and the native enzyme is that the former protein is unable to bind a monomeric lipid molecule into its 'active" site. These results indicate the significance of active site binding on the overall interaction between the enzyme and lipid-water interfaces and support the postulated ordered sequence of events in lipolysis. In the evaluation of the data described above, it has to be realised that bind:ng of PLA 2 to lipid micelles is not a simple additive process. De Araujo et al. [9] showed that insertion of PLA z into micellar nhexadecyiphosphocholine (-- C t : P N ) results in a reorganization of the lipid monomers. Whereas miceiles of C,,-PN alone consist of 155 + 3 monomers, the aggregate of PLA2 and C~t,-PN consists of two protein molecules and about 80 lipid monomers. Because of these variations in stoichiometry and taking into account the large differences in the overall binding conslants for complexes of PLA_, with enantiomeric inhibitor micelles, we wanted to investigate in this study the composition of these inhibitor-protein complexes by several independent techniques. In addition, timeresolved fluorescence spectroscopy was applied to obtain more information on the interaction of the enzyme with monomerie inhibitors present in an organized lipid-water interface.

Materials and Methods Phospholipase A 2 from porcine pancreas was obtained and its activity was tested as described by Nieuwenhuizen et al. [10]. A catalytically inactive form was prepared by the reaction of PLA, with l-bromo2-octanone. The resulting octanon-PLA 2 was prepared and purified according to Verheij et al. Ill]. nHexadecylphosphocholine (C u,-PN) was synthesized as reported by Van Dam-Mieras et al. [12]. The synthesis of the optical antipodes of 2-tetradeeanoylaminohexanol- l -phosphocholine ( R- and S-C ~4-PN ) and 2-te tradecanoylamino-hexanoi-l-phosphoglycol (--R- and S-C 14-PG) is given in [4,6]. The stereoisomerie forms of 2-undecyisulfoamino-hexanoi-l-phosphocholine ( = R and S-Cz:sulfoamino-PN) and 2-undecylsulfoaminohexanol- l-phosphoglycol ( = R- and S-C,l-sulfoamiuoPG) were prepared as described previously [8]. Unless otherwise stated measurements were all performed in 25 mM Tris (pH 8.0k 20 mM CaCI,. and 150 mM NaCl.

Determination of the critical micelle concentration The critical micelle concentration (CMC) was measured by the Wilhelmy plate method as described described by Davies and Rideal [13] or by means of the soluble probe 8-anilinonaphtalene sulphon;c acid (ANS) as proposed by De Vendittis et al. [14]. ANS

was excited at 370 nm and emission was recorded at 470 nm.

Competitice &hibit!on ]'he first paper of this series [3] presented a general model for the competitive inhibition of PLA 2 at interfaces containing substrate (S1. inhibitor (1) and/or detergent (D). In the presence of mixed micelles the steady-state velocity is describe :t by the following formula rb = Kc"'" £"

I

D

(I)

if we assume that the area per molecule of inhibitor and detergent are identical, we can define R v as velocity in the presence of an inhit:itor with Kt*

=

Kin*

R~ = velocity in the presence of an inhibilor with K1* :~ K~

(2)

and a as the molar fraction of inhibitor (a = I/[I + S]). Introducing these definitions, Eqn. l can be rewritten as

to,* K,* ~- I + a Z

(3)

The slope of the straight line, R v = f (a) represents the inhibitory power Z of the inhibitor molecule tested.

Equilibrium gel filtration Equilibrium get filtration experiments were performed on FPLC (Pharmacia FPLC system; Sweden) as follows. A Superdex 75 HR 10/30 column was equilibrated with buffer (25 mM Tris (pH 8.0), 20 mM CaCI z and 150 mM NaCl). The column was calibrated u3ing the Boehringer calibration kit: cytochrome c, chymotr:psinogen, hen egg albumin, bovine serum albumin, aidolase and ferritine (molecular mass 12.5-450 kDa). Good resolution of proteins was obtained in the range of 3 to 81 kDa. The elution volume of these proteins did not change when micellar concentrations of C,,-PN were present. In order to estimate the molecular weight of the hydrated lipid-protein complexes, the column ¢¢as equilibrated with buffer containing a variable concentration of the lipid analogue under study. Samples of 100 #1 containing protein (25/~M) were dissolved in this buffer and applied to the column. The elution profile was followed by absorbance of the fractions at 280 nm and sometimes by testing the PLA z activity in the fractions. PLA 2 activitiy was measured on an eggyolk suspension in the presence of deoxycholate, as described by Nieuwenhuizen et al. [10].

~7

Light scatterblg measurements Buffer, enzyme and lipid solutions were filtered under pressure through Millipore filters (20 nm) to free them from dust particles. Lipid and PLA 2 concentrations were determined before and after filtration by phosphorus content according to Rouser et at. [15] and by absorbance at 280 rim, respectively. Protein concentrations did not change by filtration. Buffered lipid solutions decreased between 5-10% in concentration. Refractive index increments were measured with a Rayleigh interfelometer (Aus Jena, DDR) at 546 nm and 25°C and were found to be 0.131 +0.002 and 0.200 + 0.007 ml/g for C~-PN and PLA2, respectively. This !s it! good agreement with the values reported earlier [9,16]. For the lipid analogues used in this study we measured 0.134 + 0.001, 0.120 + 0.002 and 0.077 + 0.001 m l / g for both isomers of R- and S-C~4-PN, Rand S-Ctl-sulfoamino-PN, and R- and S-Cltsulfoamino-PG, respectively. Viscosity problems hampered the determination of the refractive index increment of R- and S-C~4-PG. Static light scattering measurements were performed at 546 nm and 25°C using a FICA light scattering photometer at angles between 45* and 135°. The anhydrous molecular weights were calculated from the light scattering intensity et 90' and refractive index increments using the equation for the light scattering of dilute solutions of monodisperse particles [I6-18]. Experiments were performed in the concentration range from 0 to 8 mM lipid and light-scattering intensities at 900 were corrected for pure solvent scattering. The lipid to protein molar ratios of the complexes were determined as described by de Araujo et al. [9]. Dynamic light scattering experiments were carried out using an argon-ion laser (Spectra Physics 2020; A at 488 nm) and a 128 channel correlator (Malvern 7032ce) at 25°C. The autocorrection function was fit with a single exponential function. From the resolved fluctuation decay function, diffusion coefficients are calculated. Diffusion coefficients were measured for pure enantiomeric lipid analogues, enzyme free and lipidenzyme complexes. Using the Stokes-Einstein relation a hydrodynamic radius, rn, is calculated from these diffusion coefficients.

Analytical uitracentrifi~gation Sedimentation constants for the complexes of PLA: with R. and S-C~,cPN were determined on a Beckman model E analytical ultracentrifuge equipped with electronic speed control carried out at 60090 rpm at 25°C. Double sector 12-ram cells were filled with 200 pl of a PLA 2 solution (80/zM) in the presence of the lipid analogue (200 ~M) which has a CMC value of 10 t~M. In mixed PLA2-1ipid samples, the enzyme was monitored by scanning at 280 nm. For ultracentrifugation experiments, as well as in the light scattering experi-

ments, the lipid to protein ratio was chosen to provide sufficient detergent to bind nearly all protein, avoiding a large excess of lipid micelles without bound enzyme. From the hydrodynamic parameters S (sedimentation velocity in Svedberg units) and D (diffusion constant in (mm ~ s- ~)), the molecular weight of the PLA_, containing parthc!e is estimated using the Svcdberg equation.

Steady-~h:te and thne-re~'olcedfluorescence spectroscopy Fluorescence spectra were recorded at 21"C on a modified SLM 8000 spectrofluorometer, interfaced to a Macintosh SE microcomputer. The emission polarizer was set al 55° from the vertically oriented excitation polarizer. Correction for the polarization bias was performed with an unpolarized sample (N-acety]-tryptophanamidc in buffer at 21°C). Steady-state fluorescence anisotropy was measured on the same spectro. fluorometer in the T-format mode. Excitation wavelength was set at 300 nm (l-nm bandwidth), and the fluorescence emission was collected through a cutoff filter (1 M CuSO 4, 2-cm optical path). Time-resolved fluorescence was measured on the experimental set-up of synchrotron radiation machine, Super-ACO. Total fluoresence and anisotropy decays were obtained from the polarized components Iw(t) and lvh(t). The storage ring provides a light pulse with a full width at half maximum) oi" ~ 500 ps at a frequency of 8.33 MHz for a double bunch mode. Excitation wavelength was set al 330 nm (bandwidth 5 nm) and the emission wavelength was set al 350 nm (bandwidth I0 nm). In the experimental set-up a Hammamatsu microchannel plate RI564U-06 was used in most cases. Occasionally a XP2020Q phototube was used. Data accumulation was stopped when 105 to 2-105 counts were stored in the peak channel for the total fluorescence intensity decay. The instrumental response function was automatically monitored in alternating with the parallel and perpendicular components of the polarized fluorescence decay by measuring the sample-scattering light at the emission wavelength. Anisotropy decay analysis was carried out by non-linear least-square regression. For a residue in a protein, the internal rotation is restricted. The semiangl¢ of the cone (tOmu~) of the free rotation in the subnanosecond time scale of the fluorophore transition dipole was calculated according to Kinosita et al. [19]. Data analysis of the total intensity decay was performed by the Maximum Entropy Method (MEM), as described by Livesey and Bronchon [20], Protein solutions of 46 ~tM were prepared in standard buffer. Two types of experiments were performed: the interaction of PLA 2 with micellar lipid analogues and the int~:raction of PLA~ incorporated into Ct~,-PN 'host' micelles with a single inhibitor molecule. For the direct interaction of PLA, with micellar lipid analogues (Table It) a final concentration

98 of 0.8 mM was used. To incorporate all free PLA: into 'host' micelles of C~cPN (Table l i d a concentration of 12 mM was used. To ih:estigat¢ binding of an inhibitor molecule to the active site of the enzyme, two mole equivalents inhibitor per PLA 2 were added to the system. Results Physico-chemical characterizalion of micelles The physico-chemical behaviour of aggregated lipids with well-defined chain length and polar headgroups was determined. The data ~.re presented in Table I. Both enantiomers of stereochemically pure lecithin analogues were examined separately and yielded identical data. The product analogue Ci6-PN is added for reasorJ~ o,¢ comparison. Our results are consistent with the formation of a homogeneous population of C ~,-PN micelles (157-1-4 lipid ~nonomers per micelle), and confirm the value of 155 ,'- 3 reported by de Araujo et al. [9]. In all experiments the light scattering intensity of Ct6-PN micelles showed no angle dependence (between 45° and 135°). Cu,-PN most probably forms spherical structures with a hydrodynamic radius of 35+ 1 A. The stcreoisomeric forms of the inhibitors 2-tetradecaneylamino-hexanol-l-phosphocholine ( R- and S-C 14PN) display CMC values of 10 p.M, identical to the CMC value o-~ " r.,, Furthermore similar anhydrous L ~16-rls. molecular weights are calculated for the Cu,-PN, Rand S-C 14-PN lipid micelles, indicating that the overall size of Ct6-PN, R- and S - C I : P N lipid micelles is comparable. However, in the case of R- and S-C ~4-PN we observed an angle dependence in the hydrodynamic radius, This was also found for the 2.undecylsulfoamino-hexanol-l-phosphocholines ( = R - and SCi:sulfoamino-PN). Obviously the higher CMC values of these latter lecithin analogues result in smaller lipid

aggregates (106 + 6 lipid monomers per micelle). All lecithin analogues used in this paper contain a short alkyl chain of four carbon atoms on the C-1 and a much longer (sulfo)aminoacyl chain attached to the C-2-position. This asymmetry might be the cause of some polydispersity. Also phosphoIipid analogues were examined carrying a phosphoglycol instead of a phosphocholinc headgroup. In Table I, one notices that replacing a phosphocholine by a phosphoglycol moiety has no effect on the CMC. However, significant differences are observed when comparing the lipid aggregates formed. The 2-undccylsulfoamino-hexanol-l-phosphoglycol stereoisomcrs (R- and S-Cl:sulfoamino-PG) gi'~e rise to micelles with an anhydrous molecular mz.ss of 743 kDa. Roughly 1500 lipid monomers of thest: negatively charged lipids organize into one micelle, l'he samples containing lipid aggregates of 2-tetradecanoylaminohexanol-l-phosphoglycol ( = R- and S-C14-PG) in the presence of Ca~+-ions, were very viscous, therefore, refractive index increments could not be detcrmin¢d. The dynamic light scattering data of R- and S-C ~4-PG demonstrated the presence of even larger aggregates when compared with the micelles of R- and S . C t : sulfoamino-PG. In addition the intensities of th(= !ight scattering at 90° were non4incar with concentration. Because the organization of r'G-inhibitor micelles complicates the interpretation of lipid-protein interactions, wc decided tt3 focus on the phosphocholine containing analogues.

The interaction of phospholipase A 2 with micellar zwitterionic inhibitors It has been known for several years that the binding of PLA 2 to lipid micelles is not a simple additive process with one PLAz molecule binding to one mice!!e [9]. Unique species are formed that remain stable

TABLE I Physico.chemitalparamciers of t'arionsphosphofipid analogues at 25°C pH For experimentalconditions see Materials and Methods. Phosghotipid analogue # C~e-PN R. or S-C14-PN R- or S-C~:sulfoammo-PN R- or S-C14-PG R- or $-C Ii-sulfoamino'PG

CMC (pM) i0 !0 50 10 50

Anhydrous molecular mass(kDa) a 64 72 53 * 743

Lipid monomers per micelle ~ 157 146 IU6 * 1445

rh (rim) b 3.5-3.5-3.5 5.3-4.3-3.9 5.6-3.9-3.6 59.0-33.9~28.0 12.1- 12.3-12.1

The enantiomcri¢ lecithin analogueswere measured separately. Because they give similarcharacterislicsonly one of them is incorporated in the table. ' Resultsbased on static lightscatteringexperiments. b Hydrodynamicradius obtained from dynamic light scattering intensitydecays at 45°, 90°, 135°, * No reliable determination possible, due to formation of very large micelles(with polydispersity).

99 over a considerable ~oncentration range of enzyme and lipid. These species need to be studied using several techniques. Therefore molecular weights of lipid-protein complexes were estimated by gel filtration experiments. Fig. 1 shows the elution patterns of PLA 2 on a calibrated Superdex 75 HR column. The formation of a lipid-protein complex was investigated by gradually increasing the lipid to protein molar ratio from 1 to 16 in the equilibration buffer. In the left part of Fig. 1, the interaction of PLA 2 with R-CI4-PN is clcpicted. With one mole equivalent we already observed high molecular mass complexes. By increasing the R-Ct4-PN to protein ratio, the dynamic balance between the several populations shifts towards one final complex of 7 4 '= 2 kDa. An 8-fold molar excess of lipid over end, me is sufficient to complex all PLA 2. The right part of Fig. 1 shows the results obtained with the S-isomer of this analogue. This lipid lacks the proper stereochemical conformation for active site binding. In the presence of S.C,4-PN the protein eluted at a position similar to that in the absence of lipid analogues (-- 15 _+ 1 kDa). In the same concentration range as used for the R-iso-

UE

16.0

t

7.9

3.9

0

5

Fig. 1, A schematic representation of the elution patterns of porcine pancreatic PLA: on a Superdex 75 FIR column in the presence of different inhibitorconcentrations. In the left part of the figure the interaction of the enzymewith R-CI4-PNand in the right part the interaction with S-C|4-PN is illustrated. Samples of 25 pM PLA: were applied to the column. The lipid/protein ratio (L/E) in the sample is presented next to the elation patterns. The arrows in the left part of the figure indicate the position of peak obtained in the presence of S-CI4-PN under identical condition. For experimental detail see Materialsand Methods,

mer, high molecular mass structures are not ~bserved. The importance of active site recognition for the overall binding properties of PLA 2 is also demonstrated using the catalytically inactive octanon-PLA, under identical conditions. When octanon-PLA 2 was applied to the column in the presence of either R-Ct4-PN or S-CI4-PN, a molecular mass identical to that of free PLA 2 was ubserved (data not shown) irrespective of the enantiomeric form of the inhibitor. Light scattering, sedimentation velocities and timeresolved fluorescence spectroscopy were used to verify these molecular mass estimations and to obtain more information about the composition of the lipid-protein complexes. A survey of the results is given in Table If. Again we focus on R- and S-CI4-PN and add CtePN for reasons of comparison. The fir~t two columns in Table II are combined results from gel filtration and static light scattering experiments. The molecular mass estimations of the enzyme free in solution and bound to C~-PN are ia good agreement with results published elsewhere [9]. The hydrated molecular mass of PLA 2 with Ct~-PN was 81 kDa and 50 lipid monomers are complexed per enzyme molecule. In contrast, we found a molecular mass of 74 kDa with a lipid-protein stoichiontetry of 2.5 for the R-Ct:PN-protein complex. We did not observe lipid-protein complexes when SC t4-PN was used under identical conditions. Sedimentation velocities and diffusion constants were determined by ultracentrifugation and dynamic light scattering measurements, respectively. For the enzyme free in solution we obtained a ~edimentation velocity of 2.2 S and diffusion constant of i.36- 10- "j m 2 s-I. These values correspond very well with the known molecular mass of the enzyme. At an inhibitor to protein ratio of 2.5 for R-C ~4-PN-PLA 2 we obtained a sedimentation constant of 4.4 S. The diffusion con. stant of 0.48.10- ~o m z s Tof the complex, agrees with a molecular mass of 74-76 kDa obtained by light scattering and gel filtration. Similar expe.fimen.s were also performed for the enzyme in the presence of S-Ct4-PN. The sedimentation constant of the enzyme in presence of S-C~4-PN is affected. F,:om the increasea sedimentation velocity and decreased diffusion constant we calculated a molecular mass of 21 kDa. On the. other hand the gel filtration experiments described in Fig. l, showed that high molecular weight aggregates are not formed under these conditions. Due to the presence of S-C L~-PN, the diffusion constant and sedimentation velocity are slightly perturbed and do not provide a reliable value of th:~ ,'+olecular mass of the involved species. This is not occurring in the presence of R-C t4-PN, where we obtain a homogeneous population of the high molecular mass aggregate. Subsequently, the direct interaction of PLA 2 with pure inhibitor micelles was studied using time-resolved fluorescence spectroscopy. Anisotropy decay data were

I00 TABLE I! Characterizalion of phospholipase A z free bl solution and present in lipid-protein complexes formed with phospholipid analogues For experimental conditions see Materials and Methods. Phospholipid

SISand gelfiltration

DLS and analytical ultracentrifugation TRFS

analogue

molecular

SV ~

mass

PLA 2 (free) Ct,-PN R-C j4-PN S-Cz4-PN

N ~

O~

molecular

0t~ f

15 81 74 15

50 2.5 -

2.2 n.d. 4.4 2.9

1.36 n.d. 0.48 1.12

13.7 n.d. 76 21

~m~,, ~

Competitive inhibition Z h

mass t

mass e

a

molecular

6.7 34.6 30.5 7.4

17 86 76 18.5

26 13 i2 23

1 190 1

SLS and D1S are static and dynamic lighl scattering, respectively, TRFS stands for time-resolved fluorescence spectroscopy, n.d.= not determined. Hydrated molecular mass in kDa obtained from gel filtration experiments. b Lipid-protein stoichiometry in the complex as determined by static light scattering measurements. c Sedimentation velocity in Svedberg units determined by ultracentrifugation. 0 Diffusion constant in 10- t. m : / s obtained from dynamic light scattering experiments. c Molecular mass in kDa derived from the Svedberg equation. Rotational correlation time of the hydrated particle in ns as obtained from time re~lved fluorescence measurements and taolecular mass in kDa based hereupon. g The amplitude of motion calculated according to Kinosita et al. [19]. h in first approximation the inhibRory power (Z) represents the ratio of dissociation constants of the substrate (R}-l,2-didodecanoyl-glycero-3phosphocholine and the lecithin analogue in the interface, i.e., ( K * / K i * . For details see Materials and Methods and Ref. 3.

fair agreement with those obtained by gelfiitration,

a n a l y z e d a s s u m i n g a s u m of e x p o n e n t i a l s r ( t ) = ~-~i e x p ( - t / O ~ ) , ~ is the c o n t r i b u t i o n to the a n i s o t r o p y of t h e c o r r e l a t i o n time 0~. T h e d a t a for P L A 2 f r e e in s o l u t i o n s h o w a fast m o t i o n for the indole ring at the p r o t e i n s u r f a c e a n d a slow m o t i o n d e s c r i b i n g t h e t u m bling rate o f the protein. T h e i n t e r n a l m o t i o n o f t h e Trp-3 r e s i d u e ( d a t a n o t s h o w n ) is slowed d o w n drastically w h e n t h e e n z y m e b i n d s to a l i p i d - w a t e r interface. T h e v a l u e s for t h e l o n g c o r r e l a t i o n :lines are c o n s i d e r ably i n c r e a s e d f r o m 6.7 ns for t h e f r e e e n z y m e to 34.6 a n d 30.5 ns for the e n z y m e c o m p l e x e d with C~,-PN a n d R-CI4-PN, respectively. T h e s e long c o r r e l a t i o n t i m e s c o r r e s p o n d w i t h a particle size o f 86 k D a for the c o m p l e x w i t h C , , - P N a n d 76 k D a w i t h R-C~4-PN, respectively. T h e m o l e c u l a r w e i g h t e s t i m a t i o n s are in

light s c a t t e r i n g a n d u l t r a c e n t r i f u g a t i o n studies. Alt h o u g h t h e w o b b l i n g a n g l e o f r o t a t i o n a l m o t i o n calculated f r o m the residual a n i s o t r o p y (mm.~) is reducecl u p o n b i n d i n g o f P L A 2 to a micellar l i p i d - w a t e r inter-

face, the mobility of the indole ring of the enzyme when present in complexes with C lc,-PN or R-CI4-PN displays similar values. In summary, the interaction of PLA 2 with R-Ct4-PN micelles resulted in a particle with an average molecular mass of 76 ~ i kDa and a protein to lipid stoichiometry of 2.5. In the same concentration range high-molecular weight aggregates of PLA 2 are not detected in the presence of the optical antipode, S-

C t4-PN,

TABLE Ill Effect of phospholipid analog,¢ binding on Jhe total intensity decay parameters of porcine phospholipase A 2 as detennined by time resoh'ed fluorescence spectroscopy at 25°C, ptl 8 a Protein with PLA~ no addition PLA, +C1~ - PN ( = host miceile) PLAt in host micelle + R - C~ - PN PLA 2 in host micelle + R - CI4-PG

ct

c,

c3

c4

¢I (ns)

~': (ns)

¢L~ (ns)

¢4 (ns)

(~'i) (ns)

0.37

0.38

0.22

0.03

0.17

0.83

3. | 3

6.27

1.26

0.07

0.20

0.73

-

0.44

1.78

3.41

-

2.88

-

0.08

0.92

-

-

!.05

3,23

-

3,06

-

0,07

0.93

-

-

1.31

3,27

-

3.1 !

The lord| in;ensi~, d~_~v was assumed: T(t)~ i. ira)el exp(- t/'ti). ¢j values are the barycenters of the lifetime class j and c i value~ are the normalized areas over each class. (~j) was calcub~ted as (Tj) ~ ~.cjr r

101

1 1 f14

.....

e

. . . . . . . .

[

. . . . . . . .

I

•

,_.8 6 ----4 E =2

0.1

1

lifetime

I0

~ v

~

.....

lo, t 1 ,o']

2

o.

|,

10

(nt)

!

b

~o,.,i 1 oSt 1 0 ° / .... , 0.1

A

1 Immm.

ii

lO

(as)

I.$104~.............. ~' 1.2 1 0 ' .

| 4.0 ~o'.

hzteraction of hzhibitors with the actil,e site of PLA2 #zcorporated bt 'host' micelles For PLA z containing a single tryptophan, the total fluorescence intensity decay is descr!bed by a discrete series of exponentials. Four lifetime classes are resolved for the enzyme free in solution. Results are shown in Fig. 2 and Table Ill. The lifetime distribution of the excited state is similar to that reported earlier [21]. The longest component contributes only to a minor extent. Large modifications of the lifetime distribution are observed upon binding of PLA, to micelles. The interaction of the enzyme with Cu,-PN micelles results in a more homogeneous fluorescence decay of Trp-3. The barycenter value of the c.~ lifetime class ( ~ 3 ns) becomes dominating (the contribution increases from 22% for the unliganded protein to 73% in the presence of micellar C I(,-PN). The two other remaining lifetime classes display higher values. If we subsequently add a 2-fold molar excess of R-Ct4-PN over PLA2 to this system, the mixed lipid-water interface consists of one molecule R-C,4-PN per 130 Cu,-PN molecules, in this ternary system the excited state lifetime distribution becomes even simpler. The major component, the 3 ns class, now rcpresents more than 90% of the total intensity decay. If we use the optical antipode S-CI4-PN instead of R-C,4-PN, the lifetime distribution remains identical with that of pure CurPN micelles. The highly reproducible lifetime data (see Table l i d show an almost single conformation of Trp-3 in the presence of inhibitors with the proper stereochemical conformation (R-C ~cPN or R-C t4-PG)incorporated in a miccllar interface (C u,-PN). The presence of the inhibitor is demonstrated by the increase in the average barycenter of all lifetime classes, (~'i)- The present finding~ indicate that binding of the inhibitor to the active site of PLA 2 leads to an additional perturbation of the tryptophan-3 environment. Discussion

o.o

"zoe~

0.1

1

(M)

10

1.5

One of the most intriguing aspects of lipolysis is the considerable rate enhancement of lipolytic enzymes when ac:ing at interfaces. The kinetic model we previously described [1] consists basically of two successive steps. The first step is a reversible penetration of the enzyme into an interface. Binding to the interface

~1.o ~o4. E I o.o

1~.

0.1

1

10

Fig. 2. MEM-rccovcrcd lifetime distribution of excited states of PLA~: (a) protein free in ~lution; (b) protein in the presence of 12 mM CI6-PN; (c) protein in the presence of an inert matrix of Cu,-PN molecules and 2 tool equivalent R-Cz4-PN: (d) protein in the presence of an inert matrix of C,,-PN molecules and 2 tool equivalent $-Cz4-PN. The experimental conditions are described under Materials and Methods.

102 could result in a different conformational state of the enzyme [12]. Subsequently the enzyme binds a single substrate molecule, thereby forming an interracial 'Michaelis-Menten complex'. Similar events are seen in the inhibition of these enzymes. The most potent competitive inhibitors possess the R-configuration and are only effective after incorporation into an organized lipid-water interface. Micelles composed of enantiomeric phospholipid analogues are ideal to study interfacial activation, since the optical antipodes have identical physicochemical properties, in this study, we investigated the interaction of PLA 2 with pure inhibitor micelles and with single inhibitor molecules diluted in a micellar interface consisting of inert CI~,-PN molecules. We examined the physicochemical characteristics of both stereoisomers of four different phospholipid analogues: the phosphoglycol derivatives (R- and S-C,4PG, R- and S-Cu-sulfoamino-PG) and the phosphocholine derivatives (R- and S-CI4-PN, R- and S-CHsulfoamino-PN). The lipid aggregates formed by phosphatidyiglycol analogues contain at least 1500 lipid monomers. Based on steric arguments the structures of these micelles can not be spherical, in contrast, the light scattering measurements of aqueous solutions of Cn(,-PN showed a homogeneous population of micelles composed of 157 lipid monomers in agreement with the spherical micelles composed of 1S5 Ctd-PN monomers reported by de Araujo et al. [9]. The phosphocholine analogues examined in this study form micelles of similar size comprising 100-150 lipid monomers. The anhydrous molecular mass of the two stereoisom~rs of both lecithin analogues (R- and SCi,-PN, R- and S-Cn-sulfoamino-PN) is also close to that of the product analogue C,,-PN. However, for these lecithin analogues we observed an angle dependence in the h~drodynamic radii pointing to polydispev.~ty in the solutions. A similar observation is also reported by Atwood et aL [22]. These authors studied u-he micellar structu~-s formed by lecithins containing a buqa'oyl- and an oteoyl chain. They interpreted their viscosity increment and light scattering data to indicate that these micell~ ~ elongated structures. We used various direct binding techniques to investigate the interaction of PLA 2 with micelles of Cto-PN and R- .rod S-Ct,-PN. We confirm the results reported by de Araujo ¢t al. [9] who showed that insertion o[ the [I.A 2 into a C,,-PN micelle results in a reorganization of the lipid momm~ts. C,,-FN micelles consist of 155 + 3 monomers. After envyme penetration the aggregate of PLA 2 and C,,-PN, has been described to comprise two protein molecules and 80 lipid monomers. R= and S-Ct4-PN micgllcs contain 146 + 14 monomers. With the R-L~ncr a comple~ of 75 ± I kDa was found with a protein to lipid stoichiometry of 2.5, If we assume 10 H 2 0 molecules per ledthin monomer [9, 23]

and no hydration of the protein, the final complex could contain 4.8 enzyme molecules. However, if we take a hydration ratio of 0.4 g H 2 0 / g dry protein [21] into account, we calculate 3.4 enzyme molecules in the final comples. Assuming an average hydration ratio of 0.2 g [-][20/g dry protein, the final complex consists of four ~nzyme molecules and ten inhibitor molecules. The calculated molecular mass of this aggregate, being 74 kDa, was observed by several techniques. The high number of enzyme molecules in the aggregate inevitably leads to protein-protein interactions and may lead to partial dehydration. Under the same conditions where high-molecular complexes are formed between R-Ct4-PN and PLA 2, no high-molecular weight aggregates are observed with S-Ct4-PN. The stereoisomers R-Ct4-PN and S-Ct4-PN show an enormous difference in their affinity to the active site of PLA~, a Z value of 190 for R- and a Z value of 1 for S-C~4-PN, respectively. Since the physicochemical properties of S- and R-Ct4-PN micelles are identical, we demonstrate here that active site binding plays an important role in the overall binding of PEA 2 to lipid-water interfaces. This is supported by the finding that the 1-bromo-2-octanone inhibited phospholipase was unable to discrimina:e between the the lipid analogues. The present study enables us to extend our model concerning the interaction of PLA 2 with lipid-water interfaces. A schematic representation is depicted in Fig. 3. The first step in the interaction of PLA 2 and a lipid-water interface, is a rather nonspecific, reversible adsorption step: E ~ E* [8]. Once present in the interface, the enzyme can bind a single inhibitor molecule, forming the two-dimensional E* 1 complex. However, for inhibitors with elevated Z values the presence of E* I leads to the formation of complexes of the type E~ly. In a previous paggr of this series [8], we measured the two-dimensional Michaelis-Menten constants for several inhibitors. The present results implicate that in fact apparent values may have been obtained. The determined dissociation constant, might be composed of K* and art aggregation constant (Karl), which cannot be measured independently.

ExIy E*I

--"-'4" 1 +

E*+

E

S ~

E*S

producls

Fig. 3. Proposed rnodcl for the action of PLA 2 (E) at an interface in the presence of a zompetit~c inhibitor (|) and/or substrale molecule (S}_ This model is an extension of the previous model given in Re£ 3.

103

We showed that potent competitive inhibitors possessing the R-configuration are only effective after incorporation into an organized lipid-water interface. The implications of the parallelism between ~ubstrate hydrolysis and competitive inhibition of lipolytic enzymes being only effective in the presence of interfaces is intriguing [6]. In order to explain interfacia| activation several theories have been put forward: (i) The active site of the enzyme is rigid, but in the presence of a lipid-water interface the hydrolysis of phospholipid molecules is enhanced due to a facilitated diffusion of the lipid molecules into the act.:ve site. [24]. (ii) The active site is flexible and in the presence of a lipidwater interface, the enzyme presumably undergoes a conformational change, giving rise to an improved active site [1,25]. (iii) The enzyme is monomeric in solution also in this theory, but binding to a lipid-water interface is thought to induce aggregation of the PLA 2 with a concomitant increase in activity. A first kinetic indication for the formation of PLA 2 dimers of Naja naja naje snake venom PLA2 was reported by Roberts et ai. [26] who explained their data by assuming that in the dimer one PLA, monomer is involved in substrate binding and the other in catalysis. Substrate-induced aggregation was also reported for N. melanoleuca PLA2 [27]. This enzyme forms in the presence of monomeric n-tridecylphosphocholine a lipid-protein complex consisting of four protein and about 36 lipid molecules and it probably also aggregates in the presence of diheptanoyl lecithin. With negatively charged substrates porcine PLA2 is able to build up comparable aggregates. Acting on monomeric S-n-alkanoylglycoisulfates the active enzyme complex was shown to consist of four enzyme molecules and about 30 substrate monomers [28,29]. Due to the fact that complexes of a highly active enzyme with substrate molecules are per definition short-living species, direct proof for their existence is difficult to obtain. Our data show that in the presence of inhibitors PLA 2 forms such complexes and it is proposed in this paper that the inhibition of PLA 2 might well be explained in line with the third mechanism. Enzyme aggregation of the type E~I* seems t.o be dependent of tight, specific binding of an inhibitor molecule to the active site of PLA 2. In answering the question whether the aggregation process is specific or due to the presence of a hydrophobic chain in the active site, it is pertinent to note that a covalently bound hydrophobic chain as it is present in octanon-PLA 2 does not lead to enzyme aggregation. Th~s suggests that the process under study is not an aspecific hydrophobic packing of en~me molecules, but depends on the correct orientation of the substrate or inhibitor molecule. Whether the aggregation, that we observed with inhibitors, is indeed essential for the mechanism of activation remains difficult to decide. It should be realized that if the ~ctiva-

tion faoor is very high, it is sufficient when only a small part of the enwme is activated to cause a large kinetic effect. Irrespective of the fact which of the three mechanisms presented above, is the more important one~ it has to be stressed that these theories are not mutaally exclusive and could operate simultaneously.

,Acknowledgements We thank R. Dijkman for the synthesis and purification of lipid analogues. A.D. is grateful to Dr. P. Weisenborn for revising the manuscript. This research was carried out with the financiai support of the Bridge Programme of the European Economic Community and fellowship support was obtained from Unilever Research Laboratory (Vlaardingen).

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