Enhanced activity and altered specificity of phospholipase A2 by deletion of a surface loop.

Protein engineering and x-ray crystallography have been used to study the role of a surface loop that is present in pancreatic phospholipases but is absent in snake venom phospholipases. Removal of residues 62 to 66 from porcine pancreatic phospholipase A2 does not change the binding constant for micelles significantly, but it improves catalytic activity up to 16 times on micellar (zwitterionic) lecithin substrates. In contrast, the decrease in activity on negatively charged substrates is greater than fourfold. A crystallographic study of the mutant enzyme shows that the region of the deletion has a well-defined structure that differs from the structure of the wild-type enzyme. No structural changes in the active site of the enzyme were detected.

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.

Phospholipase A2 isoenzyme from porcine pancreas. Purification and some properties.

Porcine pancreas synthesizes a prephospholipase A2 which occurs in a 5 : 95 ratio compared with the more abundant zymogen of the same enzyme (phosphatide-acylhydrolase; EC 3.1.1.4). These two prephospholipases could be well separated by CM-cellulose chromatography. Both the active and the zymogen form of the isoenzyme were isolated and purified. The activation peptides of both prephospholipases appeared to be identical, while the active enzymes showed a few interesting differences. The most striking differences were the loss of one histidine and one methionine in the isoenzyme, corresponding to residues 24 and 27, respectively, in alpha-phospholipase A2. The positional and stereo specificity of both enzymes are the same, but the specific activity of the beta-phospholipase A2 is lower. The molecular weight of the isoenzyme was estimated to be about 14 000, while the isoelectric points were 5.1 and 5.9 for the isoprecursor and active isoenzyme, respectively.

Structure of 1-acyl lysophosphatidylcholine and fatty acid complex in bilayers.

The phase transition characteristics of bilayers formed in a codispersion of 1-acyl lysophosphatidylcholine and a fatty acid depend on the chain length of both the components and on the pH of the aqueous medium. Incorporation of cholesterol as a third component abolishes the transition. It is suggested that acyl chain interactions between fatty acid and 1-acyl lysophosphatidylcholine molecules in their aqueous codispersions are maximized by close-packing such that the acyl chains of both molecules are aligned parallel to each other and the carboxyl group is located in the vicinity of the 2-hydroxyl group of lysophosphatidylcholine. The shape and size of a functional dimer thus formed are similar but not identical to those of 1,2-diacyl phosphatidylcholine. Several predictions arising from this suggestion, including phase separation in codispersions of fatty acid + 1-acyl lysophosphatidylcholine + diacyl phosphatidylcholine, are experimentally confirmed.

Activation of phospholipase A2 by freshly added lysophospholipids.

Reaction progress curves for the hydrolysis of dimyristoylphosphatidylcholine by pig pancreatic phospholipase A2 exhibits a latency phase. Addition of 1-palmitoyllysophosphatidylcholine to the preformed vesicles reduces the latency phase and enhances the binding of phospholipase A2 to the vesicles. In contrast, the binary codispersions prepared from diacylphospholipids premixed with lysophosphatidylcholine do not exhibit such enhanced susceptibility to the phospholipase. This effect appears to be due to organizational defects created by asymmetrical incorporation of lysophospholipid molecules into the outer monolayer of the vesicles, and the action of phospholipase is not observed when the additive is equilibrated in both the monolayers of the vesicles.

The complete primary structure of phospholipase A2 from human pancreas.

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.

Studies on the role of methionine in porcine pancreatic phospholipase A2.

The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.

Competitive inhibition of lipolytic enzymes. III. Some acylamino analogues of phospholipids are potent competitive inhibitors of porcine pancreatic phospholipase A2.

Competitive inhibition of porcine pancreatic phospholipase A2 was studied in mixed-micellar systems composed of long- and medium-chain substrates, potential inhibitors and detergents. A number of positional and stereoisomeric monoacylamino, acyloxyglycerophospholipids were investigated for their inhibitory properties, using as substrates the corresponding diacyl-sn-glycero-3-phospholipids possessing the same polar headgroup and identical acyl chain lengths. Based on a kinetic model applicable to water-insoluble inhibitors (see accompanying paper I), which allows a quantitative comparison of the inhibitory power (Z) of the various phospholipid analogues, the following results were obtained: Substitution of a single acylester bond in a diacylglycerophospholipid by an acylamino group can transform the substrate molecule in a potent competitive inhibitor. This property is acquired only when this substitution occurs on the phospholipase-susceptible ester bond of the substrate. If the acylamino group replaces an ester bond which cannot be attacked by the highly positional and stereospecific phospholipase, the resulting molecule binds with similar affinity to the active site of the enzyme as the parent substrate molecule. Because of its positional and stereospecificity, this so-called inhibitory 'amide effect' suggests that these inhibitors behave as substrate-derived analogues. The inhibitory 'amide-effect' observed with several medium- and long-chain monoacyloxy-, monoacylamino-deoxyglycerophosphatides is completely lost upon specific alkaline hydrolysis of the single acylester bond. Reesterification of the free glycerol OH group in these lysoacylaminophosphoglycerides, even with an acetyl residue, restores the inhibitory properties. These observations indicate that specific binding of phospholipids to the active site of pancreatic phospholipase A2, requires the presence of two chains in substrate or inhibitor structure and suggest that those results obtained with lysophospholipids and single-chain analogues may be questionable.

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.

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.

A Fourier transform infrared spectroscopic (FTIR) study of porcine and bovine pancreatic phospholipase A2 and their interaction with substrate analogues and a transition-state inhibitor.

Fourier transform infrared spectroscopy has been used to investigate the secondary structure of porcine and bovine pancreatic phospholipase A2 (PLA2) and the zymogen of porcine PLA2, prophospholipase A2 (proPLA2), in both H2O and D2O media. Detailed qualitative analysis was made of these proteins using second derivative and deconvolution techniques. Quantitative studies of the proteins in solution made using Factor Analysis gave average values of 54% alpha-helix, 15% beta-sheet and 23% beta-turns. These values agree well with the secondary structures deduced from previous studies of single crystals using X-ray techniques. No significant differences in secondary structure were observed for porcine pancreatic (pro)phospholipase A2 in the presence or absence of Ca2+ ions, or in the temperature range 10-45 degrees C. The binding of the non-degradable phospholipid analogue, n-alkylphosphocholine, in monomeric form produced no significant difference in the secondary structure of either enzyme. Conformational differences were, however, observed between the enzyme lyophilised in a solid film and in aqueous solution. The change is probably due to the formation of beta-sheet upon hydration, coupled with a loss of random structures. Conformational differences in both porcine and bovine pancreatic PLA2 were observed on binding to n-alkylphosphocholine micelles. This change may be due to a small increase in the alpha-helical structure and a decrease in the beta-sheet, and/or possibly beta-turn content. Similar conformational changes were observed for the interaction of porcine and bovine PLA2 with the substrate analogue inhibitor 1-heptanoyl-2-heptanoylamino-2-deoxy-sn-glycero-3-phospho glycol in micellar form.

The primary structure of phospholipase A2 from porcine pancreas. A reinvestigation.

The primary structure of porcine pancreatic phospholipase A2 (EC 3.1.1.4) has been reinvestigated. A number of modifications have been introduced including the addition of a 7th disulfide bridge. The structure which is presented here shows a high degree of homology with the amino acid sequence of snake venom and horse pancreas phospholipase A2.

Isolation and properties of prophospholipase A2 and phospholipase A2 from horse pancreas and horse pancreatic juice.

Two phospholipases A2 (EC 3.1.1.4) with different isoelectric points have been isolated from horse pancreas in high yield (880 mg/kg tissue). From pancreatic juice the more acidic species was isolated as the sole phospholipase A2. Upon tryptic activation the zymogens release a hepta- and pentapeptide, respectively from the N-terminal part of the protein giving rise to the formation of one single enzyme with a specific activity higher than that of pancreatic phospholipases A2 from other mammalian species. Horse phospholipase A2 differs from the porcine and bovine enzymes with respect to amino acid composition and kinetic properties. The sequence of the first 41 amino acid residues at the N-terminus has been determined by automatic Edman degradation.

Origin of the latency phase during the action of phospholipase A2 on unmodified phosphatidylcholine vesicles.

The reaction progress curve for the action of pig-pancreatic phospholipase A2 on dimyristoylphosphatidylcholine vesicles is characterized under a variety of conditions. The factors that regulate the rate of hydrolysis during the presteady-state phase determine the latency period. The results demonstrate that the accelerated hydrolysis following the latency phase of the reaction progress curve is due to the product-assisted binding of the enzyme to the substrate bilayer by chaning the number of bindings sites and therefore the binding equilibrium. A critical mole fraction of products appears to be formed in the substrate bilayers before the steady-state phase of hydrolysis begins. The latency phase shows a minimum at the phase-transition temperature of the substrate vesicles; however, we did not observe a significant binding of the enzyme to pure substrate bilayers even at the phase-transition temperature. The rate of binding of the enzyme is found to be fast and the rate of desorption of the bound enzyme is very slow compared to the latency phase. The rate of redistribution of products between substrate bilayers is rather slow. These observations demonstrate that during the latency phase of the action of phospholipase A2, a critical mole fraction of products is formed in the substrate bilayer.

The amino acid sequence of the phospholipase A2 isoenzyme from porcine pancreas.

The primary structure of porcine pancreatic isophospholipase A2 (EC 3.1.1.4) has been investigated. The sequence of procine isophospholipase differs from the sequence of porcine phospholipasy by four substitutions; viz. Ala12 leads to Thr; His17 leads to Asp leads to; Met20 leads to Leu and Glu71 leads to Asn.

Phase properties of the aqueous dispersions of n-octadecylphosphocholine.

Properties of the aqueous dispersions of n-octadecylphosphocholine are examined by differential scanning calorimetry, fluorescence depolarization, light scattering, 31P-NMR, pig pancreatic phospholipase A2 binding, and X-ray diffraction. On heating, these dispersions exhibit a sharp lamellar to micelle transition at 20.5 degrees C. The lamellar phase consists of frozen (gel-state) alkyl chains which do not bind phospholipase A2. The kinetics of the transition are asymmetric: the micelle to lamellar transition is very slow and the lamellar to micelle transition is fast. It is suggested that the lamellar phase is a frozen chain bilayer in which the chains interdigitate.

Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors.

It is now becoming clear from the abundant lipolytic enzyme literature that any meaningful interpretation of inhibition data has to take into account the kinetics of enzyme action at the lipid/water interface. We attempt in the present paper to provide a kinetic model applicable to water-insoluble competitive inhibitors, in order to quantitatively compare the results obtained at several laboratories. We derived kinetic equations applicable to the pre-steady state as well as steady state. By measuring the inhibitory power, as described in the present paper, it is possible to obtain a normalized estimation of the relative efficiency of various potential inhibitors. Furthermore, with the kinetic treatment developed here, it is possible to make quantitative comparisons with the same inhibitor placed under various physico-chemical situations, i.e., micellar or monolayer states.

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.

The importance of glycine-30 for enzymatic activity of phospholipase A2.

The nearly conserved glycine-30 in porcine pancreatic phospholipase A2 has been replaced by serine. The resulting mutant G30S was expressed in Escherichia coli, purified and characterized. The mutation caused a significant drop in enzymatic activity towards monomeric and aggregated substrates, but had a limited effect on substrate binding. In contrast the affinity for calcium ions, the essential cofactor, was reduced 10-fold. The reduced enzymatic activity is attributed to a reduced stabilization of the transition state. The results are discussed in view of naturally occurring inactive phospholipase A2 homologues from snake venom.

Ajoene prevents fat digestion by human gastric lipase in vitro.

Human gastric lipase (HGL) is a sulfhydryl enzyme which has been shown by Gargouri et al. (Gargouri, Y., Moreau, H., Piéroni, G. and Verger, R. (1988) J. Biol. Chem. 263, 2159-2162) to be inhibited by hydrophobic disulfides. Since HGL is involved in the digestion and absorption of dietary fats we have investigated in vitro the ability of ajoene, a natural disulfide to inactivate HGL. Ajoene is derived from ethanolic garlic extracts. The finding that ajoene inactivates HGL is consistent with the fact that it is reactive towards sulfhydryl compounds and also corroborates previous reports on the ability of garlic to lower triacylglycerol blood levels. These data may explain the age-old Mediterranean and Oriental belief in the 'blood-thinning' effects of garlic on a molecular and physiological basis.