Active Site Of Phospholipase A2 Research Articles

Enzyme Inhibition Activity Both in vitro and in silico Screening of Triphala Plant Extracts on Phospholipase A2

Objectives: The present work was aimed to determine the effect on the Phospholipase A2 (PLA2) enzyme and conduct its antioxidant nature, phytochemical analyses of the Triphala compound’s extract, an Ayurvedic Rasayana made from Terminalia chebula, Emblica officinalis and Terminalia bellirica and also, the molecular docking study. Methods: Twelve Triphala compounds are extracted from Gas chromatography-mass spectrometry, Phytochemical study, the Triphala’s inhibitory effect on antioxidant property and PLA2 assay. To study each compound’s degree by docking software like iGEMDOCK, Auto dock Vina, and overlapping on the PLA2 enzymes like Naja naja, Human sPLA2, Bee and Scorpion venoms phospholipase. Results: Phytochemical screening revealed the presence of phenolics, alkaloids, saponins, flavonoids and tannins. Methanolic extract of Triphala fruit powder also showed good scavenging ability compared to the superoxide. IC50 value of seed powder extract is 23.12 μg/ml whereas standard, i.e. superoxide is at 32.66 μg/ml, respectively. IC50 scavenging activity was 31.18. μg/ml and 32.66 μg/ml standard, respectively. Moreover, it shows a better interaction of bioactive compounds with the PLA2 enzyme. Morin (3,5,7,2’,5’-Pentahydroxyflavone) 9,10 anthraquinone was found to be a better inhibitor among the compounds, with a binding energy -82.6534K. cal/mol, -73.9662K.cal/mol respectively and hydrogen bonding with PRO- 61 and ARG-59, the primary amino acid residues at the active site of PLA2. Conclusion: The study will allow the Triphala compounds that contain lead in oncological therapy and other inflammatory diseases to be improved.

Characterization of a Novel Thrombin-like Enzyme, Globlase from the Venom of Gloydius blomhoffii (Japanese Mamushi)

Aims: To elucidate the coagulation mechanisms of a novel clotting factor isolated from Gloydius blomhoffii venom, its hydrolytic activity on various substrates were examined. Furthermore the primary structure was determined and compared with the other snake venom components.
 Methodology: A thrombin-like enzyme was isolated from the crude venom of G. blomhoffii by DE52 Cellulose and CM52 Cellulose column chromatography. Enzyme activity was measured by using synthetic substrates (arginine esters, MCA-substrates and 3-(Acyloxy)-4-nitrobenzoic acid). Effect on fibrinogen was detected with bovine and human fibrinogen. Isoelectric point and molecular mass were measured by polyacrylamide gel electrophoresis and MALDI-TOF-MS. Amino acid sequence was decided with a protein sequencer by analyzing enzymatically cleaved peptides.
 Results: A clotting factor was found to be homologous as indicated by a single band on SDS-PAGE, and the final preparation was named as globlase. Molecular mass of this enzyme was determined to be 13,876.36 Da and the isoelectric point was 8.8. Globlase showed arginine ester hydrolytic activity, and specificity for substrates of thrombin. Proteolytic activity and phospholipase A2 (PLA2) activity were not detected. Complete amino acid sequence analysis indicated that the primary structure of globlase is similar to PLA2. However the aspartic acid which exists in the active site of PLA2 was found to be substituted by glutamine.
 Conclusion: It was shown in our current investigation that globlase is a novel thrombin-like enzyme isolated from G. blomhoffii venom. It was revealed that this enzyme had structure unlike the serine-protease such as the other thrombin-like enzymes.

Computational and in vitro insights on snake venom phospholipase A2 inhibitor of phytocompound ikshusterol3-O-glucoside of Clematis gouriana Roxb. ex DC.

Ikshusterol3-O-glucoside was isolated from Clematis gouriana Roxb. ex DC. root. A structure of the isolated compound was determined on the basis of various spectroscopic interpretations (UV, NMR, FTIR, and GC-MS-EI). This structure was submitted in the PubChem compound database (SID 249494133). SID 249494133 was carried out by density functional theory calculation to observe the chemical stability and electrostatic potential of this compound. The absorption, distribution, metabolism, and excretion property of this compound was predicted to evaluate the drug likeness and toxicity. In addition, molecular docking, quantum polarized ligand docking, prime MMGBSA calculation, and induced fit docking were performed to predict the binding status of SID 249494133 with the active site of phospholipase A2 (PLA2) (PDB ID: 1A3D). The stability of the compound in the active site of PLA2 was carried out using molecular dynamics simulation. Further, the anti-venom activity of the compound was assessed using the PLA2 assay against Naja naja (Indian cobra) crude venom. The results strongly show that Ikshusterol3-O-glucoside has a potent snake-venom neutralizing capacity and it might be a potential molecule for the therapeutic treatment for snakebites.

The Role of Calcium on the Active Site of Snake Venom Phospholipase A2: Molecular Dynamics Simulations

Snake venoms are rich in phospholipase A2 (PLA2) and their hydrolysis of cell membrane phospholipids explains the role of the enzyme in venom toxicity. Calcium is known to plays important role at the active site of PLA2 during catalysis. In this study, molecular dynamics simulations of free PLA2 and calcium bound PLA2 were carried out using GROMACS 4.5.5 to evaluate the role of calcium in PLA2 catalysis. The results showed that calcium induced formation of helical structures between Arg62 - Lys66, Asn107 - Tyr111 and Asp114 - Cys119 in PLA2 which with time disappeared through the formation and opening of loops. Calcium induced atomistic movements and conformational changes in snake venom PLA2 which led to the formation of a widened cleft at the active site of calcium bound PLA2 when compared with free PLA2. This could lead to a better binding and accommodation of substrate, thus enhancing catalysis. This study confirms the role of calcium towards the action of PLA2 in snake venom toxicity and could provide useful information for the design of small molecules that can function as PLA2 inhibitors.

High-affinity selective inhibitor against phospholipase A2 (PLA2): a computational study

Phospholipase A2 (PLA2) is the most abundant protein found in snake venom. PLA2 induces a variety of pharmacological effects such as neurotoxicity, myotoxicity and cardiotoxicity as well as anticoagulant, hemolytic, anti-platelet, hypertensive, hemorrhagic and edema inducing effects. In this study, the three dimensional structure of PLA2 of Naja sputatrix (Malayan spitting cobra) was modeled by I-TASSER, SWISS-MODEL, PRIME and MODELLER programs. The best model was selected based on overall stereo-chemical quality. Further, molecular dynamics simulation was performed to know the stability of the modeled protein using Gromacs software. Average structure was generated during the simulation period of 10 ns. High throughput virtual screening was employed through different databases (Asinex, Hit finder, Maybridge, TOSLab and ZINC databases) against PLA2. The top seven compounds were selected based on the docking score and free energy binding calculations. These compounds were analyzed by quantum polarized ligand docking, induced fit docking and density functional theory calculation. Furthermore, the stability of lead molecules in the active site of PLA2 was employed by MD simulation. The results show that selected lead molecules were highly stable in the active site of PLA2.

Substituted thiobenzoic acid S-benzyl esters as potential inhibitors of a snake venom phospholipase A2: Synthesis, spectroscopic and computational studies

4-Chlorothiobenzoic acid S-benzyl ester (I), 3-nitrothiobenzoic acid S-benzyl ester (II), 4-nitrothiobenzoic acid S-benzyl ester (III) and 4-methylthiobenzoic acid S-benzyl ester (IV) were prepared and characterized by 1H and 13C NMR, Mass spectrometry and IR spectroscopy. Quantum chemical calculations were performed with Gaussian 09 to calculate the geometric parameters and vibrational spectra. Phospholipase A2 (PLA2) was purified from Crotalus durissus cumanensis venom by molecular exclusion chromatography, followed by reverse phase-high performance liquid chromatography. Two studies of the inhibition of phospholipase A2 activity were performed using phosphatidilcholine and 4-nitro-3-octanoyloxybenzoic acid as substrates, in both cases compound II showed the best inhibitory ability, with 74.89% and 69.91% of inhibition, respectively. Average percentage of inhibition was 52.49%. Molecular docking was carried out with Autodock Vina using as ligands the minimized structures of compounds (I–IV) and as protein PLA2 (PDB code 2QOG). The results suggest that compounds I–IV could interact with His48 at the active site of PLA2. In addition, all compounds showed Van der Waals interactions with residues from hydrophobic channel of the enzyme. This interaction would impede normal catalysis cycle of the PLA2.

Quercetin modulates activities of Taiwan cobra phospholipase A2 via its effects on membrane structure and membrane-bound mode of phospholipase A2

The goal of the present study is to elucidate the mechanism of quercetin on modulating Naja naja atra phospholipase A2 (PLA2) activities. Sphingomyelin inhibited PLA2 enzymatic activity and membrane-damaging activity against egg yolk phosphatidylcholine (EYPC), while cholesterol and quercetin abrogated the sphingomeyelin inhibitory effect. Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity toward EYPC/sphingomyelin/cholesterol vesicles. Both cholesterol and quercetin increased detergent resistance and reduced membrane fluidity of EYPC/sphingomyelin vesicles. Quercetin reduced detergent insolubility but increased ordered lipid packing of EYPC/sphingomyelin/cholesterol vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that quercetin altered the membrane-bound mode of PLA2 differently upon absorption onto the membrane bilayers of different lipid compositions. However, 8-anilinonaphthalene sulphonate-binding assay revealed that quercetin marginally affected the interaction between active site of PLA2 with phospholipid vesicles. Collectively, our data indicate that membrane-inserted quercetin modulates PLA2 interfacial activity and membrane-damaging activity via its effects on membrane structure and membrane-bound mode of PLA2.

Structural Biology of Recombinant Bovine Pancreatic Phospholipase A2 and its Inhibitor Complexes

The enzyme phospholipase A2 catalyzes the cleavage of the sn-2 acyl ester bond of phospholipids, leading to the production of free fatty acids and lysophospholipids, which leads to many inflammatory disorders. In view of its pharmaceutical interest, three phospholipase A2 + inhibitor (namely, (i) L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine, Transition State Analogue, (ii) 1-Hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol, MJ33 and (iii) p-methoxybenzoic acid, anisic acid) complex structures have already been solved and analysed, using the data obtained from X-ray diffraction. These structures provide insight on the mode of binding of the inhibitor molecules at the active site of phospholipase A2. The knowledge of the active site geometry in these inhibitor bound structures, yield valuable information in the design of more useful therapeutic agents. This report reviews only the inhibitor bound recombinant bovine pancreatic phospholipase A2 structures solved using X-ray crystallography.

Crystal structure of phospholipase A2 complex with the hydrolysis products of platelet activating factor: equilibrium binding of fatty acid and lysophospholipid-ether at the active site may be mutually exclusive.

We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their occupancies set to zero in the refined model due to disorder. Together, the crystallographic and equilibrium binding results with the two products show that the simultaneous binding of both the products in a single active site is not favored.

Cover Picture: ChemBioChem 7/2002

The cover picture shows the petrosaspongiolide M molecule (PM), a marine natural product with potent anti-inflammatory activity, docked into the active site of phospholipase A2 (PLA2). The pharmacophoric γ-hydroxybutenolide ring of PM lies in close proximity to the N terminus of the enzyme. Further details can be found in the article by Gomez Paloma and co-workers on p 664 ff.

Extraction of Lipids from Phospholipid Membranes by Steered Molecular Dynamics

Steered molecular dynamics (SMD) simulations were employed to investigate the extraction step of the lipid head group cleavage reaction by human synovial phospholipase A2 (PLA2) by pulling a lipid molecule from a monolayer of dilauroyl-phosphatidyl-ethanolamin lipids into the active site of PLA2 and into the aqueous phase. The results of the simulations were compared to draw inferences about the forces that stabilize the lipids in the membrane and to understand the mechanism of lipid extraction by PLA2.

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2.

In order to address the mechanism whereby Ca2+ wad crucial for the manifestation of the enzymatic activity of phospholipase A2 (PLA2), four divalent cations were used to assess their influences on the catalytic activity and the fine structures of Naja naja atra PLA2. It was found that substitution of Mg2+ or Sr2+ for Ca2+ in the substrate solution caused a decrease in the PLA2 activity to 77.5% or 54.5%, respectively, of that in the presence of Ca2+. However, no PLA2 activity was observed with the addition of Ba2+. With the exception of Mg2+, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA2 markedly increased with the binding of cations to PLA2. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate and p-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca2+, Sr2+, and Ba2+, but not by Mg2+. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba2+ > Sr2+ > Ca2+ > Mg2+, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA2 and that Tyr-3, Lys-6, and Tyr-63 of PLA2 are involved in the binding with the substrate, suggest that the binding of Ca2+ to PLA2 induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg2+ or the bigger atomic radii with Sr2+ and Ba2+ might render the conformation improperly rearranged after their binding to PLA2 molecule.

The essentiality of His-47 and the N-terminal region for the binding of 8-anilinonaphthalene-1-sulfonate with Taiwan cobra phospholipase A2.

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to Naja naja atra phospholipase A2 (PLA2) as well as the enhancement of ANS fluorescence of the PLA2-ANS complex decreased with increasing pH in a pH range from 3 to 9. These pH-dependent curves can be well interpreted as the perturbation of an ionizable group with pK value of 5.8, which was assigned as His-47 in the active site of PLA2. The ionizable group with pK 5.8 was no longer observed after methylation of His-47, supporting the idea that the pH dependence of ANS binding arose from an electrostatic interaction between His-47 and the bound ANS. Removal of the N-terminal octapeptide of PLA2 caused a precipitous drop in the capability of PLA2 for binding with ANS and enhancing ANS fluorescence, reflecting that the integrity of the N-terminal region was essential for maintaining the hydrophobic character of the ANS-binding site. However, the nonpolarity of the ANS-binding site in the N-terminus-removed derivative was still partially retained at low pH, but was completely lost at high pH. Evidently, the N-terminal region plays a more crucial role in ANS binding at high pH than at low pH. These results indicate that hydrophobic interaction as well as electrostatic interaction are involved in the binding of ANS to PLA2, and that the relative contributions of both interactions in ANS fluorescence enhancement may be different under different pH.

Conformation of micellar phospholipid bound to the active site of phospholipase A2.

Transferred NOE techniques have been used to determine the structure of phospholipid analogues bound to the active site of cobra venom phospholipase A2 (PLA2). These experiments were carried out on PLA2 with a substrate analogue which serves as an inhibitor, 1-(hexylthio)-2-(nonanoylamino)-1,2-dideoxy-sn-glycero-3-pho sphocholine (PC9). Because this inhibitor binds tightly to the enzyme and forms micelles at millimolar concentrations, experiments could be carried out to determine the conformation of the inhibitor when bound to the enzyme at the lipid-water interface. NOEs of the micellar lipid develop inefficiently in the absence of enzyme. NOESY experiments in the presence of PLA2 were used to determine the inhibitor structure and conformation when bound to the enzyme. The inhibitor adopts an active site conformation in which the end of the sn-2 chain is within 5 A of the alpha-methylene protons of the sn-1 chain. However, NOE cross-peaks in the experiments indicate that the backbone conformation of the bound lipid is different from that of a shorter chain lipid which forms monomers [Plesniak et al. (1993) Biochemistry 32, 5009-5016].

Binding of active site directed ligands to phospholipase A2: Implications on the molecular constraints and catalytic mechanism

Molecular constraints for the localization of active site directed ligands (competitive inhibitors and substrates) in the active site of phospholipase A2 (PLA2) are characterized. Structure activity relationships with known inhibitors suggest that the head : group interactions dominate the selectivity as well as a substantial part of the affinity. The ab initio fitting of the amide ligands in the active site was carried out to characterize the head group interactions. Based on a systematic coordinate space search, formamide is docked with known experimental constraints such as coordination of the carbonyl group to Ca2+ and hydrogen bond between amide nitrogen and ND1 of His48. An optimal position for a bound water molecule is identified and its significance for the catalytic mechanism is postulated. Unlike the traditional ''pseudo-triad'' mechanism, the ''Ca-coordinatedoxyanion'' mechanism proposed here invokes activation of the catalytic water to form the oxyanion in the coordination sphere of calcium. As it attacks the carbonyl carbon of the ester, a near-tetrahedral intermediate is formed. As the second proton of the catalytic water is abstracted by the ester oxygen, its reorientation and simultaneous cleavage form hydrogen bond with ND1 of His48. In this mechanism of esterolysis, a catalytic role for the water co-ordinated to Ca2+ is recognised.

Inactivation of human pleural fluid phospholipase A2 by dioscin

The natural product, spirostanol glycoside dioscin, was shown to directly inactivate human pleural fluid phospholipase A2 (PLA2). Inactivation was dose, and time dependent. The IC50 was estimated at 18 μM and virtually complete inactivation of the enzyme occurred at 50 μM. Using Michaelis-Menten kinetics, dioscin inactivated the enzyme by a competitive inhibitory manner, the apparentKi value was 6.9×10−4M. Reversibility was studied directly by dialysis method, the inhibition was reversible. Addition of excess Ca2+ concentration up to 8 mM did not antagonize the inhibitory activity of dioscin. Inactivation of several kinds of PLA2 by dioscin, showed a broad range of PLA2 specificity. These data suggest that inactivation of PLA2 by dioscin is due to interaction with the active site of PLA2 and may be a useful adjunct in the theraphy of inflammatory diseases.

Design and synthesis of some substrate analogue inhibitors of phospholipase A2 and investigations by NMR and molecular modeling into the binding interactions in the enzyme-inhibitor complex.

A series of substrate analogue inhibitors of pancreatic phospholipase A2 has been designed and synthesized. The compounds were tested in a novel dual-screening system based on parallel assays with monomeric and micellar substrates. Intermolecular nuclear Overhauser effects between vinylic protons on one inhibitor and identified active site residues on the bovine pancreatic enzyme have been observed in solution NMR studies of the enzyme-inhibitor complex. It can be deduced from both the biochemical results and the NMR data that the mode of interaction between this type of inhibitor and the active site of phospholipase A2 is essentially the same, irrespective of the presence or absence of an aggregated phospholipid surface. A model of the binding between the enzyme and inhibitor which incorporates the two-dimensional NMR data has been developed. The model can account for the activity of modified inhibitor structures and can be extrapolated to an assessment of the mode of binding of the natural substrate itself.

Competitive inhibition of lipolytic enzymes VIII: Inhibitor-induced aggregation of porcine pancreatic phospholipase A 2

Several 2-acylaminophospholipid analogues have been demonstrated to behave as potent competitive inhibitors of porcine pancreatic phospholipase A2 (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 observed only when incorporated into a micellar 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-tetradecyl-amino-hexanol-1-phosphocholine (R-C14-PN and S-C14-PN). Upon equilibrium gel filtration of the enzyme (monomeric molecular mass = 14 kDa) on calibrated Superdex columns running in micellar solutions of R-C14-PN, the phospholipase eluted as a lipid-protein complex of 74 kDa. Under identical conditions, micellar solutions of S-C14-PN did not give rise to high-molecular mass aggregates and the enzyme eluted at its normal 14 kDa position. Light scattering experiments, ultrasedimentation and time-resolved fluorescence spectroscopy studies confirmed the formation of a high-molecular mass aggregate between enzyme and R-C14-PN micelles. The ultimate complex was shown to consist of four protein and about ten inhibitor molecules. Using time-resolved fluorescence spectroscopy the interaction was studied between the active site of phospholipase A2 and R-C14-PN molecules, both incorporated in an inert lipid matrix.

Active-site-directed specific competitive inhibitors of phospholipase A2: novel transition-state analogs

More than 100 amphiphilic phosphoesters, possible tetrahedral transition-state analogues capable of coordinating to the calcium ion at the active site of phospholipase A2, were designed, synthesized, and tested as inhibitors for the hydrolysis of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol vesicles in the scooting mode. This assay system permits the study of structurally diverse inhibitors with phospholipase A2S from different sources, and it is not perturbed by factors that change the quality of the interface. As a prototype, 1-hexadecyl-3-trifluoroethylglycero-2-phosphomethanol (MJ33) was investigated in detail. Only the (S)-(+) analogue of MJ33 is inhibitory, and it is as effective as the sn-2 phosphonate or the sn-2 amide analogues of sn-3 phospholipids. The inhibitory potencies of the various phosphoesters depended strongly on the stereochemical and structural features, and the mole fractions of inhibitors required for 50% inhibition, X1(50), ranged from more than 1 to less than 0.001 mole fraction. The affinity of certain inhibitors for enzymes from different sources differed by more than 200-fold. The inhibitors protected the catalytic site residue His-48 from alkylation in the presence of calcium but not barium as expected if the formation of the EI complex is supported only by calcium. The equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface was correlated with the XI(50) values, which were different if the inhibition was monitored in the pseudo-zero-order or the first-order region of the progress curve. These results show that the inhibitors described here interfered only with the catalytic turnover by phospholipase A2's bound to the interface, their binding to the enzyme occurred through calcium, and the inhibitors did not have any effect on the dissociation of the enzyme bound to the interface.

Catalytic Ca2+-binding site of pancreatic phospholipase A2: laser-induced Eu3+ luminescence study.

7F0----5D0 excitation spectroscopy of Eu3+ has been used to study the catalytic Ca2+-binding site of pancreatic phospholipases A2. Eu3+ binds competitively with Ca2+ to the enzyme with retention of about 5% of the activity found with Ca2+. The dissociation constants for the Eu3+-enzyme complexes of bovine phospholipase A2 and porcine isophospholipase A2 are 0.22 mM and 0.16 mM, respectively. Results obtained with the porcine phospholipase A2 at neutral pH indicate aggregation of this enzyme at protein concentrations above 0.18 mM. The Eu3+ bound at the catalytic site of pancreatic phospholipase A2 is coordinated to four or five water molecules, which, in conjunction with binding constant data, suggests the involvement of two or three protein ligands. Addition of a monomeric substrate analogue to the enzyme-Eu3+ complex results in the loss of an additional water molecule from the first coordination sphere of the bound Eu3+. This result suggests an interaction between the negative charge of the polar head group of the substrate analogue and the Eu3+. Binding of the enzyme-Eu3+ complex to micelles results in a nearly complete dehydration of the Eu3+ bound to the catalytic center. In the phospholipase A2-Eu3+-micelle complex, only one H2O molecule is coordinated to Eu3+. This dehydration at the active site of phospholipase A2 in the protein-lipid complex can be an important reason for the enhanced activity of this enzyme at lipid-water interfaces.