Ruud Dijkman

Interaction of phospholipase A2 and phospholipid bilayers.

Binding of phospholipase A2 from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PC14) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC14 alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of pig-pancreatic enzyme to vesicles of the diether-PC14 could not be detected even after long incubation (up to 24 H) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.

Phosphonate analogues of triacylglycerols are potent inhibitors of lipase.

1,2-Dioctylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates, with alkyl being methyl or octyl, were synthesised and tested as irreversible inhibitors of cutinase from Fusarium solani pisi and Staphylococcus hyicus lipase. Rapid inactivation of these enzymes occurred with a concomitant release of one mole of p-nitrophenol per mole of enzyme. With both lipases a higher reactivity was observed when the alkyl substituent on the phosphonate is a methyl rather than an octyl chain. Both lipases are highly selective for the chirality of these compounds at glycerol and at phosphorus. Rapid inactivation at an inhibitor concentration of 0.1 mol% in 100 mM NaTDOC (t 1/2 < 60 min.) occurred when the glycerol moiety had the (R) configuration, while inhibitors of the (S) configuration react 4-10-fold more slowly. The isomer with the p-nitrophenyl octylphosphonate attached to the secondary hydroxyl group of glycerol hardly inhibited (t 1/2 > 1 day) the lipases. These results reflect the known positional- and stereopreference of these enzymes which preferentially release the fatty acid at sn-3 of natural triacylglycerols. The enzymes appeared to be even more selective for the chirality at phosphorus, the differences in reactivity of the faster and slower reacting isomers being as high as about 250-fold for the methylphosphonates and about 60-fold for the octylphosphonates. These phosphonates can be regarded as true active site-directed inhibitors. The inhibited enzymes can be considered as analogues of the tetrahedral intermediate in the acylation step that occurs during triacylglycerol hydrolysis.

Competitive inhibition of lipolytic enzymes. IV. Structural details of acylamino phospholipid analogues important for the potent inhibitory effects on pancreatic phospholipase A2

1-Acyl-2(R)-acylamino phospholipids are effective competitive inhibitors of porcine pancreatic phospholipase A2 (EC 3.1.1.4, Bonsen et al. (1972) Biochim. Biophys. Acta 270, 364-382). By systematically varying the substituent at C-1 and the acyl chain length at C-2, a series of phospholipid analogues was obtained for which the inhibitory power was determined in a detergent-containing and occasionally also in a detergent-free micellar substrate system. The recently proposed kinetic model applicable to water-insoluble inhibitors (Ransac et al. (1990) Biochim. Biophys. Acta 1043, 57-66) allowed a quantitative comparison of the inhibitory power Z of the various substrate analogues. The most powerful inhibitors of the enzyme were found to possess the general 2-(R)-structure: (formula; see text) Using as substrate (R)-1,2-didodecanoylglycero-3-phosphocholine in mixed micelles with sodium taurodeoxycholate, the inhibitor molecule with m = 4 and n = 11 showed a Z-value of 15,000. This implies an affinity of the inhibitor for the active site of the enzyme higher than 4 orders of magnitude stronger as compared with the substrate molecule. Slightly higher and lower m-values resulted in a sharp drop of the inhibitory power, which suggests that the enzyme must possess a rather short, but well-defined hydrophobic binding pocket for the C-1 alkyl chain. Variation of n (keeping m = 2 constant) resulted in inhibitors with nearly equal Z-values for n = 11, 13 and 15. Most probably the binding cleft on the enzyme for the C-2 acylamino chain is longer, more losely constructed and contributing less to the overall binding energy. Several members of the 2-acylamino phospholipids are water-soluble and possess relatively high critical micelle concentrations. Their inhibitory power could be tested not only in micellar substrate dispersions but also in assay systems where both the inhibitor and substrate are molecularly dispersed. It appeared that these water-soluble phospholipid analogues are effective inhibitors of the enzyme only after incorporation into an organized substrate/water interface. In contrast, in molecularly dispersed substrate solutions the same molecules have completely lost their inhibitory power. These observations support our kinetic model of lipolysis and interfacial inhibition.

Competitive inhibition of lipolytic enzymes. II. Preparation of ‘monoacylamino’ phospholipids

This paper describes the synthesis of a number of phosphatidylcholines and phosphatidylglycols, in which one fatty acyl ester group is replaced by an acylamino function. The phospholipids, both of the alpha- and beta-type, are prepared in racemic and enantiomeric pure forms.

Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor.

SummaryThe three-dimensional structure of porcine pancreatic PLA2 (PLA2), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 ϕ torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA2 and PLA2 in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal α-helix. Whereas free in solution Ala1, Leu2 and Trp3 are disordered, with the α-amino group of Ala1 pointing out into the solvent, in the ternary complex these residues have an α-helical conformation with the α-amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA2. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA2 in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA2.

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A2 (PLA2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257]. Both the mutant platelet PLA2 and the human pancreatic PLA2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14-16 carbon atoms on position two. Because the platelet PLA2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.

Competitive inhibition of lipolytic enzymes. IX: A comparative study on the inhibition of pancreatic phospholipases A2 from different sources by (R)-2-acylamino phospholipid analogues

The inhibitory power (Z) of a number of (R)-1-alkyl-2-acylamino phospholipid analogues was determined for three mammalian phospholipases A2 from pig, ox and horse pancreas. All three enzymes display a clear preference for anionic (phosphoglycol) inhibitors over the zwitterionic (phosphocholine) derivatives; this effect is most pronounced for the bovine enzyme. Upon variation of the 1-alkyl chain length, the bovine and equine phospholipases, like the porcine enzyme in previous studies, show an optimum in Z for a six-carbon alkyl group. The introduction of a double bond in the 2-acylamino group generally improves the inhibitory power as compared with a fully saturated acyl chain. For the horse enzyme, the presence of an (R)-2-undecenoylamino group in the phosphocholine- and phosphoglycol-containing inhibitors resulted in affinities which are nearly 4 and 5 orders of magnitude higher, respectively, than for the substrate molecule. Direct determination of the dissociation constant Ki* of several inhibitors incorporated in a host lipid/water interface of non-inhibitory n-octadecenylphosphocholine micelles, was performed by ultraviolet difference spectroscopy. The progressive binding of a single inhibitor molecule into the active site of the three enzymes was followed quantitatively by an increasing tyrosine perturbation. With moderately strong competitive inhibitors (Z values ranging from about 50 to 10,000), quantitative values for Ki* were obtained. Extrapolation of the experimentally found linear relationship between Z and 1/Ki* yields predicted Ki* numbers for the much stronger inhibitors with Z values between 10,000 and 100,000.

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 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 micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers 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-inhibitory 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 large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.

Competitive inhibition of lipolytic enzymes. X. Further delineation of the active site of pancreatic phospholipases A2 from pig, ox and horse by comparing the inhibitory power of a number of (R)-2-acylamino phospholipid analogues

Two series of (R)-phospholipid analogues, each containing a n-propyl group at the C-1 position and various acylamino functions at the C-2 position have been synthesized and their inhibitory properties towards three mammalian pancreatic phospholipases A2 have been determined. The members of the first series of analogues all contained the zwitter-ionic phosphocholine headgroup which in the second series was replaced by the anionic phosphoglycol function. In the saturated 2-acylamino phospholipids the length of the acyl chain ranged from 8 to 18 carbon atoms. The unsaturated 2-acylamino analogues possessed a chain length of 11 or 18 carbon atoms and contained one, two, three or four double bonds. For inhibitors with a saturated acylamino group, the phospholipases A2 from pig, ox and horse show a sharp optimum in inhibitory power Z for an acyl chain length of 10 carbon atoms. The inhibitory behaviour of the unsaturated acylamino analogues is more complex: both the zwitter-ionic and the anionic inhibitors demonstrate an increase in Z with an increasing number of cis-double bonds but the degree of improvement is dependent on the position of the double bonds. Subsequently the influence of polar groups at carbon position 12 of the dodecanoylamino phospholipids on Z was analyzed. Substitution of the terminal methyl group by an OH-function lowers the inhibitory potency of the three enzymes by a factor of 4 to 5 both in the phosphocholine and phosphoglycol series. Replacement of the methyl group by potentially charged functions (-NH2, -COOH) resulted in a complete loss of inhibitory properties. Blocking of the amino group and carboxyl function by t-butyloxycarbonylation and esterification, respectively, fully restored the inhibitory power. Finally we investigated how changes in the polar headgroup and the presence of aromatic rings at the C-1 or C-2 position influenced the inhibitory potency of the analogues.

Competitive inhibition of lipolytic enzymes. XI. Estimation of the interfacial dissociation constants of porcine pancreatic phospholipase A2 for substrate and inhibitor in the absence of detergents.

Based on the strong inhibitory properties of (R)-2-decanoylamino-octanol-1-phosphocholine and its phosphoglycol analogue for porcine pancreatic phospholipase A2, the corresponding 2-decanoyloxy derivatives have been synthesised in both enantiomeric forms and their substrate properties for the enzyme were analysed. The high aqueous solubility in the absence of detergents, combined with low critical micelle concentrations of both the amide- and ester phospholipids allowed the estimation of the interfacial dissociation constants of the enzyme-substrate and enzyme-inhibitor complexes by kinetic and direct binding techniques.