73
Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phos~ho~ipase A, with micellar li~i~/water interfaces of competitive inhibitors A,M, Th. 3, Deveer ‘, P.A. Franken b, R. Dijkman b, 1. Meeldijk b, M.R. Egmond a, H.M. Verheij b, R. Verger ’ and C.H, de Haas ’ Ii Unilervr Research, t/(rmrdinxen ~Net~er~~~ds~,’ ~e~rt~~~t
of Enzymology and Pratein Engineering, C.B.L.E., Utreclzt ~~etherl~nds) and (’ C. N. R. S, C. 8. MS, ~~~eil~e (France)
(Received 31 October 1991)
Key words: Pancreatic phosphol~p~~~ A,; competitive i~~~bi&ioff:Ultraviolet difference absorption spectroscopy; Z-Acylami~~-phosphoiipid
In a recent series of kinetic studies (De Haas et al. (1990f Biochim. Biophys. Acta 1046,249-257 and references therein) we have demonstrated that synthetic ~~)-ph~spholip~d analogues containing a 2-acylam~no~roup instead of the 2-acyloxy function found in natural phosphoIipids, behave as strong c~mpctitive inh~bjtors of porcine pancreatic phosphol~pase A, (PLA,). We also showed that these analogues strongly bind to the active site of the enzyme but onIy after their incorporation into a miceliar substrate/water interface. In the present study we investigated the interaction of native PLA, and of an inactive PLA, in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and miceilar lipid/water interfaces probably consists of a Low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the tR)-co~fjguration in its active site, whereas the ~~~-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial ph~spholipaseinhibitor compfex is three orders of magnitude lower for miceiles composed of the (RI-isomer than those of the N-isomer. The modified PLA, still adsorbs to micellar ~~pid/water interfaces but cannot bind either of the two ~~antiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitor phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the targe difference in overall dissociation constants of the complexes between enzyme and micelies of (R&and (~)-inhibitors. With the use of PLA, mutants in which each time a singte tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.
Kinetic analysis of water-soluble secreted Iipolytic enzymes such as lipases and pbospholip~ses, is complicated because these enzymes exert their highest activity on substrates organized in multimoiecu~ar aggregates. In an attempt to analyze such a heterogeneous catalysis Verger et al. proposed in 1973 a general model [l]
Correspondence: G.H. de Haas, ~~p~~rnent of ~~zyrnolo~ and Protein Engineering, C.B.L.E., Tram III, Paduataan 8, 3584 CH Utrecht, Netherlands.
that is a~pliGabie to iipolysis at various kinds of interfaces. According to this model, the iipo~yt~c enzyme (E) being highly water-soluble, is present in the aqueous phase. The first interactj~~ between the li~~lyt~c enzyme E and the physically separated substrate/water interface requires a reversible interaction between the components. Once in the interface the enzyme (E*) can bind in a subsequent step a single substrate molecule in its active site forming a Michaelis-Menten complex E*S. This second eq~iiibrium is characterized by a ho-dimensional dissociation constant K,*. Experimentali~ the totaf amount of interface can be easily varied and enough substrate can be added as to bind
74
all enzyme to the interface (all E * E”). Determination of the two-dimensional K,* is more complicated since the two-dimensional substrate concentration is not easily varied over a wide range. In fact the two-dimensional concentration of the substrate can only be varied by the addition of neutral surface diluters as has been extensively discussed before [2,3]. Limited information could be obtained, so far, on the magnitude of the two-dimensional dissociation constant K,*. In the case of pancreatic and venom PLA, acting on micellar zwitterionic lecithins there are indications that K,* is much larger than the two-dimensional substrate concentration S [2-41. This implies that the initial adsorption step (E + E*) should be hardly influenced by the interfacia1 equilibrium. Therefore, it is not amazing that kinetically determined dissociation constants of these enzymes acting on different substrates and dissociation constants that are obtained by direct binding studies with non-hydrolysable substrate analogues are al1 rather similar and in the milhmolar range. Recently Ransac et al. [5] derived the kinetic equations describing the action of lipolytic enzymes on a substrate in the presence of a detergent and a competitive inhibitor. In a series of papers [5-IO] the kinetic behavior of a number of 2-acylamino lecithin analogues which behave as potent competitive inhibitors of porcine pancreatic PLA z was described. Incorporated into a lecithin/water interface, they eagerly bind to the active site of the enzyme E* and very low mole fractions of inhibitor are sufficient to block hydrolysis of the substrate. In the present study the interaction between porcine pancreatic PLA, and these inhibitors, both as pure micelles and incorporated into inert carrier micelles, has been investigated by direct binding techniques. Experimental evidence will be described supporting for the first time the two-step kinetic model for lipolysis. Materials
and Methods
Phospholipase A, from porcine pancreas was obtained as described previously [ll]. PLA, inactivated by reaction with I-bromo-2-octanone (octanon-PLA,) was prepared and purified according to Verheij et al. [12]. Mutant PLA, in which tyrosine-69 has been replaced by phenylalanine (Y69F) was obtained by sitedirected mutagenesis [13]. The preparation of PLA, lacking the surface loop 62-66 (A-PLA,) has been described previously [14]. Derived from A-PLA, two additional mutant phospholipases [I57 were obtained in which either tyrosine-52 or tyrosine-73 was replaced by phenylalanine (AY52F and AY73F). n-Hexadecylphosphocholine (C ,,-PN) was synthesized as reported by Van Dam-Mieras et al. [16]. The stereoisomeric forms of 2-dodecanoyiamino-hexano~1-phosphocholine (R-C ,2-PN; S-C ,,-PN) and of 2-te-
tradecanoylamino-hexanol-l-phosphocholine (R-C,,PN; S-C,,-PN) were prepared as described previously [7,91. The synthesis of CR)- and (.?I-Ztetradecanoylamino-hexanol-1-phosphoglycol (R-C ,,-PC; S-C ,,-PGI is given in [9]. The stereoisomers of 2-undecylsulfonylamino-hexanol-1-phosphocholine (R-C,,-sulfoaminoPN; S-C,,-sulfoamino-PN) were prepared in a similar way starting from (R)- and (S)-norleucinemethyl ester and undecylsuifonyibromide [ 171. [LY]~= t 23.11” and -23.05” for the CR)- and t.S)-isomer, respectively tC, 10 in H ,O); NMR-characterization: 360 MHz ’ H-NMR (CDCI,-MeOH = 1: 1): 6, 0.9 (6 H, 2 x U-I,), 1.3 (22 H, alkyl), 1.X (2 H, PCH,), 3.0 (2 H, c&HZ), 3.2 (9 H, NKH,),), 3.4 (1 H, CH), 3.6 (2 H, CHIN), 3.9 (2 H, CH ,OP), 4.3 (2 H, POCH *I. The buffer solution used throughout in this study consisted of 25 mM Tris-HCI, 20 mM Ca”+, 150 mM NaCl (pH 8.0). Determination of critical micelle concer2tratiorz
The critical micelle concentration (CM0 at 25°C of the various phospholipid analogues was measured by the Wilhelmy plate method as described by Davies and Rideal [18] or by means of the soluble fluorescent probe Sanilinonaphthalene sulfonic acid CANS) as proposed by De Vendittis et al. [l!?]. ANS was excited at 370 nm and emission was recorded at 470 nm. Competitice inhibition
The inhibition of phospholipase A, at pH 8 by the stereo isomers of the various acyl amino substrate analogues was determined by the pH stat method as described before (671. The substrate used in the inhibition studies was (RI-1,2-didodecanoyl-glycero-3-phosphocholine and the sum of substrate and inhibitor concentrations was kept constant at 3 mM at a constant detergent concentration of 3 mM taurodeoxycholate. The analysis of the inhibition data of phosphohpase A, by competitive inhibitors in the presence of mixed micelles composed of phospholipids and detergents has been elaborated extensively by Ransac et al. [5]. These authors derived the following equation: Z = (K,:/Ki* - l)/ CK,” + 11, where K,* and Ki* are the two-dimensional dissociation constants for substrate and inhibitor, respectively. In those cases where K,,,* z=-Ki* and with all enzyme in the interface, the inhibitory power Z approximates the ratio of the dissociation constants of the substrate (RI-1,2-didode~anoyi-glycero-3-phosphocholine and the lecithin analogue in the interface, i.e., K,*/K,*. Ultraviolet absorption
difference
spectroscopy
Difference spectra were recorded by using an Aminto Model DW 2-a spectrophotometer equipped with a MIDAN-data analyzer. The data analyzer was used to store the reference difference spectrum. The wave-
75 length range was 235-385 nm with 5 data points per nm resolution. Spectra were recorded by direct automatic subtraction of the stored spectrum from the actually measured one at 1 rim/s wavelength sweep by using noise suppression (slow response). A set of quartz tandem cuvettes was used (2 X 1 cm light path), containing standard buffer and a buffered enzyme solution in the left and right compartments of either cuvette, respectively. After storage of the reference difference spectrum, equal volumes of a lipid solution (10 ~1 or more) were added to the enzyme in cuvette A and to buffer in cuvette B, while the same volume of buffer was added to the enzyme in cuvette B. The mixtures were magnetically stirred for several minutes. The solutions were not stirred during recording of the actual difference spectrum. The observed difference signal (Qobbd) was routinely taken from the value of the absorption maximum relative to the baseline. Binding parameters were obtained as previously described by De Araujo et al. [20]. Calculations were performed on a desk top Apple II computer. A program for non-linear regression analysis, based on the method of Fletcher and Powell [21] involving a combination of the steepest decent and Gauss-Newton regression methods, was used. Data input consists of the values for total enzyme concentrations (E,), total lipid (monomer> concentration CL,) and the corresponding observed signal, Qobbd. The unweighed data are fitted to the equation:
K,=
x
where K, represents the dissociation constant of the lipid-protein complex, N the number of lipid molecules per enzyme molecule in the complex and x the concentration of that complex. The above equation, a quadratic function in X, is solved for x (yielding only one physically possible root) by using initial estimated values for K, and N. A calculated signal, Qcalcd, is then obtained from the relation Qcalcd= C. x where C is the molar ultraviolet difference extinction coefficient. Its estimated value is given as input to the regression program. The program searches for the minimum of the unweighted summed squared difference 12” (Q0t,sd-Qca,cd)21over all n data points. When a minimum is found, the parameters K,, N and C are given as output, together with their variances and covariances from which standard deviations and correlation coefficients are calculated. In those cases where the used model does not give separate N and K, values, a weak complex is formed such that the following situation arises: N. K, = (E, - xXL, - N .x)/x, where N *x . Moreover, the maximum ultraviolet difference absorption with this mutant is observed at a much higher wavelength (294 nm) as compared to the other Tyr-mutants. This behavior is in agreement with previous reports from our laboratory [13, 24-261 and from others 127-291 showing that the OH group of Tyr-69 strongly interacts with one of the oxygen atoms of the phosphate moiety of the inhibitor. The foregoing results demonstrate the high preference of the active site of PLA, for the CR)-enantiomers of the 2-acylaminophospholipid analogues as compared with the (Dantipodes. Taking into account the well-known stereospecific properties of the enzyme this outcome was not unexpected. It should be emphasized, however, that beside the stereo configuration of the ligand the specific properties of the amide bond adjacent to the C-O-P linkage, are of paramount importance for high affinity to the active site to PLA,. This could be demonstrated by comparing the interaction of PLA, with the following lecithin analogues:
H-C-NH-CO-C,,H,, Hz-C-0-PO.O--CH,-CH,-N+(CH,), O_
(H, OH)
(R)-2-dodecanoylamino-hexanol-lphosphocholine
CH, (AH,), H-C-NH.SO,-C,,H,, H,-C-O-PO~O-CH,-CH,-N+(CH3)R 0(R)-2-undecylsulfonylamino-hexanol-lphosphocholine
(H, OH)
80 TABLE V Physico-chemical parameters for binding of micellar kcithin analogucs to porcine pancreatic phosp~olipas~ A, determined by ultra&iet difference spectmsiwpy
FOFexperimental conditions see Materials and Methods. Lecithin analogue ”
CMC (@MI
Ch (MT’.crn-‘)
N.&J&’ (PM)
Zd
tRkC,,-amino-W ~~~-C~~-sulf5arn~n~P~
10 10
llfxf llho
10 1050
170
1
”
Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phos~ho~ipase A, with micellar li~i~/water interfaces of competitive inhibitors A,M, Th. 3, Deveer ‘, P.A. Franken b, R. Dijkman b, 1. Meeldijk b, M.R. Egmond a, H.M. Verheij b, R. Verger ’ and C.H, de Haas ’ Ii Unilervr Research, t/(rmrdinxen ~Net~er~~~ds~,’ ~e~rt~~~t
of Enzymology and Pratein Engineering, C.B.L.E., Utreclzt ~~etherl~nds) and (’ C. N. R. S, C. 8. MS, ~~~eil~e (France)
(Received 31 October 1991)
Key words: Pancreatic phosphol~p~~~ A,; competitive i~~~bi&ioff:Ultraviolet difference absorption spectroscopy; Z-Acylami~~-phosphoiipid
In a recent series of kinetic studies (De Haas et al. (1990f Biochim. Biophys. Acta 1046,249-257 and references therein) we have demonstrated that synthetic ~~)-ph~spholip~d analogues containing a 2-acylam~no~roup instead of the 2-acyloxy function found in natural phosphoIipids, behave as strong c~mpctitive inh~bjtors of porcine pancreatic phosphol~pase A, (PLA,). We also showed that these analogues strongly bind to the active site of the enzyme but onIy after their incorporation into a miceliar substrate/water interface. In the present study we investigated the interaction of native PLA, and of an inactive PLA, in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and miceilar lipid/water interfaces probably consists of a Low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the tR)-co~fjguration in its active site, whereas the ~~~-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial ph~spholipaseinhibitor compfex is three orders of magnitude lower for miceiles composed of the (RI-isomer than those of the N-isomer. The modified PLA, still adsorbs to micellar ~~pid/water interfaces but cannot bind either of the two ~~antiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitor phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the targe difference in overall dissociation constants of the complexes between enzyme and micelies of (R&and (~)-inhibitors. With the use of PLA, mutants in which each time a singte tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.
Kinetic analysis of water-soluble secreted Iipolytic enzymes such as lipases and pbospholip~ses, is complicated because these enzymes exert their highest activity on substrates organized in multimoiecu~ar aggregates. In an attempt to analyze such a heterogeneous catalysis Verger et al. proposed in 1973 a general model [l]
Correspondence: G.H. de Haas, ~~p~~rnent of ~~zyrnolo~ and Protein Engineering, C.B.L.E., Tram III, Paduataan 8, 3584 CH Utrecht, Netherlands.
that is a~pliGabie to iipolysis at various kinds of interfaces. According to this model, the iipo~yt~c enzyme (E) being highly water-soluble, is present in the aqueous phase. The first interactj~~ between the li~~lyt~c enzyme E and the physically separated substrate/water interface requires a reversible interaction between the components. Once in the interface the enzyme (E*) can bind in a subsequent step a single substrate molecule in its active site forming a Michaelis-Menten complex E*S. This second eq~iiibrium is characterized by a ho-dimensional dissociation constant K,*. Experimentali~ the totaf amount of interface can be easily varied and enough substrate can be added as to bind
74
all enzyme to the interface (all E * E”). Determination of the two-dimensional K,* is more complicated since the two-dimensional substrate concentration is not easily varied over a wide range. In fact the two-dimensional concentration of the substrate can only be varied by the addition of neutral surface diluters as has been extensively discussed before [2,3]. Limited information could be obtained, so far, on the magnitude of the two-dimensional dissociation constant K,*. In the case of pancreatic and venom PLA, acting on micellar zwitterionic lecithins there are indications that K,* is much larger than the two-dimensional substrate concentration S [2-41. This implies that the initial adsorption step (E + E*) should be hardly influenced by the interfacia1 equilibrium. Therefore, it is not amazing that kinetically determined dissociation constants of these enzymes acting on different substrates and dissociation constants that are obtained by direct binding studies with non-hydrolysable substrate analogues are al1 rather similar and in the milhmolar range. Recently Ransac et al. [5] derived the kinetic equations describing the action of lipolytic enzymes on a substrate in the presence of a detergent and a competitive inhibitor. In a series of papers [5-IO] the kinetic behavior of a number of 2-acylamino lecithin analogues which behave as potent competitive inhibitors of porcine pancreatic PLA z was described. Incorporated into a lecithin/water interface, they eagerly bind to the active site of the enzyme E* and very low mole fractions of inhibitor are sufficient to block hydrolysis of the substrate. In the present study the interaction between porcine pancreatic PLA, and these inhibitors, both as pure micelles and incorporated into inert carrier micelles, has been investigated by direct binding techniques. Experimental evidence will be described supporting for the first time the two-step kinetic model for lipolysis. Materials
and Methods
Phospholipase A, from porcine pancreas was obtained as described previously [ll]. PLA, inactivated by reaction with I-bromo-2-octanone (octanon-PLA,) was prepared and purified according to Verheij et al. [12]. Mutant PLA, in which tyrosine-69 has been replaced by phenylalanine (Y69F) was obtained by sitedirected mutagenesis [13]. The preparation of PLA, lacking the surface loop 62-66 (A-PLA,) has been described previously [14]. Derived from A-PLA, two additional mutant phospholipases [I57 were obtained in which either tyrosine-52 or tyrosine-73 was replaced by phenylalanine (AY52F and AY73F). n-Hexadecylphosphocholine (C ,,-PN) was synthesized as reported by Van Dam-Mieras et al. [16]. The stereoisomeric forms of 2-dodecanoyiamino-hexano~1-phosphocholine (R-C ,2-PN; S-C ,,-PN) and of 2-te-
tradecanoylamino-hexanol-l-phosphocholine (R-C,,PN; S-C,,-PN) were prepared as described previously [7,91. The synthesis of CR)- and (.?I-Ztetradecanoylamino-hexanol-1-phosphoglycol (R-C ,,-PC; S-C ,,-PGI is given in [9]. The stereoisomers of 2-undecylsulfonylamino-hexanol-1-phosphocholine (R-C,,-sulfoaminoPN; S-C,,-sulfoamino-PN) were prepared in a similar way starting from (R)- and (S)-norleucinemethyl ester and undecylsuifonyibromide [ 171. [LY]~= t 23.11” and -23.05” for the CR)- and t.S)-isomer, respectively tC, 10 in H ,O); NMR-characterization: 360 MHz ’ H-NMR (CDCI,-MeOH = 1: 1): 6, 0.9 (6 H, 2 x U-I,), 1.3 (22 H, alkyl), 1.X (2 H, PCH,), 3.0 (2 H, c&HZ), 3.2 (9 H, NKH,),), 3.4 (1 H, CH), 3.6 (2 H, CHIN), 3.9 (2 H, CH ,OP), 4.3 (2 H, POCH *I. The buffer solution used throughout in this study consisted of 25 mM Tris-HCI, 20 mM Ca”+, 150 mM NaCl (pH 8.0). Determination of critical micelle concer2tratiorz
The critical micelle concentration (CM0 at 25°C of the various phospholipid analogues was measured by the Wilhelmy plate method as described by Davies and Rideal [18] or by means of the soluble fluorescent probe Sanilinonaphthalene sulfonic acid CANS) as proposed by De Vendittis et al. [l!?]. ANS was excited at 370 nm and emission was recorded at 470 nm. Competitice inhibition
The inhibition of phospholipase A, at pH 8 by the stereo isomers of the various acyl amino substrate analogues was determined by the pH stat method as described before (671. The substrate used in the inhibition studies was (RI-1,2-didodecanoyl-glycero-3-phosphocholine and the sum of substrate and inhibitor concentrations was kept constant at 3 mM at a constant detergent concentration of 3 mM taurodeoxycholate. The analysis of the inhibition data of phosphohpase A, by competitive inhibitors in the presence of mixed micelles composed of phospholipids and detergents has been elaborated extensively by Ransac et al. [5]. These authors derived the following equation: Z = (K,:/Ki* - l)/ CK,” + 11, where K,* and Ki* are the two-dimensional dissociation constants for substrate and inhibitor, respectively. In those cases where K,,,* z=-Ki* and with all enzyme in the interface, the inhibitory power Z approximates the ratio of the dissociation constants of the substrate (RI-1,2-didode~anoyi-glycero-3-phosphocholine and the lecithin analogue in the interface, i.e., K,*/K,*. Ultraviolet absorption
difference
spectroscopy
Difference spectra were recorded by using an Aminto Model DW 2-a spectrophotometer equipped with a MIDAN-data analyzer. The data analyzer was used to store the reference difference spectrum. The wave-
75 length range was 235-385 nm with 5 data points per nm resolution. Spectra were recorded by direct automatic subtraction of the stored spectrum from the actually measured one at 1 rim/s wavelength sweep by using noise suppression (slow response). A set of quartz tandem cuvettes was used (2 X 1 cm light path), containing standard buffer and a buffered enzyme solution in the left and right compartments of either cuvette, respectively. After storage of the reference difference spectrum, equal volumes of a lipid solution (10 ~1 or more) were added to the enzyme in cuvette A and to buffer in cuvette B, while the same volume of buffer was added to the enzyme in cuvette B. The mixtures were magnetically stirred for several minutes. The solutions were not stirred during recording of the actual difference spectrum. The observed difference signal (Qobbd) was routinely taken from the value of the absorption maximum relative to the baseline. Binding parameters were obtained as previously described by De Araujo et al. [20]. Calculations were performed on a desk top Apple II computer. A program for non-linear regression analysis, based on the method of Fletcher and Powell [21] involving a combination of the steepest decent and Gauss-Newton regression methods, was used. Data input consists of the values for total enzyme concentrations (E,), total lipid (monomer> concentration CL,) and the corresponding observed signal, Qobbd. The unweighed data are fitted to the equation:
K,=
x
where K, represents the dissociation constant of the lipid-protein complex, N the number of lipid molecules per enzyme molecule in the complex and x the concentration of that complex. The above equation, a quadratic function in X, is solved for x (yielding only one physically possible root) by using initial estimated values for K, and N. A calculated signal, Qcalcd, is then obtained from the relation Qcalcd= C. x where C is the molar ultraviolet difference extinction coefficient. Its estimated value is given as input to the regression program. The program searches for the minimum of the unweighted summed squared difference 12” (Q0t,sd-Qca,cd)21over all n data points. When a minimum is found, the parameters K,, N and C are given as output, together with their variances and covariances from which standard deviations and correlation coefficients are calculated. In those cases where the used model does not give separate N and K, values, a weak complex is formed such that the following situation arises: N. K, = (E, - xXL, - N .x)/x, where N *x . Moreover, the maximum ultraviolet difference absorption with this mutant is observed at a much higher wavelength (294 nm) as compared to the other Tyr-mutants. This behavior is in agreement with previous reports from our laboratory [13, 24-261 and from others 127-291 showing that the OH group of Tyr-69 strongly interacts with one of the oxygen atoms of the phosphate moiety of the inhibitor. The foregoing results demonstrate the high preference of the active site of PLA, for the CR)-enantiomers of the 2-acylaminophospholipid analogues as compared with the (Dantipodes. Taking into account the well-known stereospecific properties of the enzyme this outcome was not unexpected. It should be emphasized, however, that beside the stereo configuration of the ligand the specific properties of the amide bond adjacent to the C-O-P linkage, are of paramount importance for high affinity to the active site to PLA,. This could be demonstrated by comparing the interaction of PLA, with the following lecithin analogues:
H-C-NH-CO-C,,H,, Hz-C-0-PO.O--CH,-CH,-N+(CH,), O_
(H, OH)
(R)-2-dodecanoylamino-hexanol-lphosphocholine
CH, (AH,), H-C-NH.SO,-C,,H,, H,-C-O-PO~O-CH,-CH,-N+(CH3)R 0(R)-2-undecylsulfonylamino-hexanol-lphosphocholine
(H, OH)
80 TABLE V Physico-chemical parameters for binding of micellar kcithin analogucs to porcine pancreatic phosp~olipas~ A, determined by ultra&iet difference spectmsiwpy
FOFexperimental conditions see Materials and Methods. Lecithin analogue ”
CMC (@MI
Ch (MT’.crn-‘)
N.&J&’ (PM)
Zd
tRkC,,-amino-W ~~~-C~~-sulf5arn~n~P~
10 10
llfxf llho
10 1050
170
1
”
