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Keywords:

  • enzyme kinetics studies;
  • isothermal titration calorimetry;
  • n-hexadecanoic acid;
  • phospholipase A2 inhibition;
  • X-ray Crystallography

Abstract

  1. Top of page
  2. Abstract
  3. Experimental Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

Ester bond hydrolysis of membrane phospholipids by Phospholipase A2 and consequent release of fatty acids are the initiating steps of inflammation. It is proposed in this study that the inhibition of phospholipase A2 is one of the ways to control inflammation. Investigations are carried out to identify the mode of inhibition of phospholipase A2 by the n-hexadecanoic acid. It may help in designing of specific inhibitors of phospholipase A2 as anti-inflammatory agents. The enzyme kinetics study proved that n-hexadecanoic acid inhibits phospholipase A2 in a competitive manner. It was identified from the crystal structure at 2.5 Å resolution that the position of n-hexadecanoic acid is in the active site of the phospholipase A2. The binding constant and binding energy have also been calculated using Isothermal Titration Calorimetry. Also, the binding energy of n-hexadecanoic acid to phospholipase A2 was calculated by in silico method and compared with known inhibitors. It may be concluded from the structural and kinetics studies that the fatty acid, n-hexadecanoic acid, is an inhibitor of phospholipase A2, hence, an anti-inflammatory compound. The inferences from the present study validate the rigorous use of medicated oils rich in n-hexadecanoic acid for the treatment of rheumatic symptoms in the traditional medical system of India, Ayurveda.

Phospholipase A2 (PLA2, E.C.3.1.1.4) catalyzes the hydrolysis of ester bonds at the sn-2 position of membrane phospholipids and releases fatty acids, such as arachidonic acid (AA) and lysophospholipids. It is the initiating step in the formation of potent inflammatory mediators (1).

The excess production of inflammatory mediators leads to pathological conditions such as rheumatoid arthritis, bronchial asthma, ulcerative colitis, SLE, psoriasis, and Crohn’s disease. Inhibitors of PLA2 may act as anti-inflammatory agents and investigation on them may help in designing of better anti inflammatory compounds.

Phospholipase A2 is abundant in a variety of natural biological fluids such as pancreatic secretions, reptile, and arthropod venoms and in certain pathologic expressions, particularly the inflammatory exudates. PLA2 plays crucial roles in diverse cellular responses, host defense, and signal transduction. The PLA2 from different sources shows minor variations in its primary and tertiary structures. However, the catalytic functions of these PLA2 isozymes are similar (2,3).

Palmitic acid (CH3(CH2)14COOH), n-hexadecanoic acid, is a saturated fatty acid (4). Many fatty acids are known to have antibacterial and antifungal properties (5). Fatty acids can modulate immune responses by acting directly on T cells (6). The dietary, conjugated linoleic acid exerts anti-inflammatory effect by decreasing production of the inflammatory mediators such as prostaglandin E2, IL-6, IL-1β, TNFα, and nitric oxide (7). The terrestrial plant-derived n-3 fatty acid, α-linolenic acid (ALA) exhibited higher anti-inflammatory effect than the sea weed-derived n-3 fatty acid, docosahexaenoic acid (DHA) (8). The polyunsaturated fatty acids, stearidonic acid (18:4n-3), and hexadecatetraenoic acid (16:4n-3) from the sea weeds Undaria pinnatifida and Ulva pertusa respectively suppressed eicosanoid production (9). A methoxylated fatty acid, 7-methoxy-9-methylhexadeca-4, 8-dienoic acid (MMHDA), isolated from the brown sea weed Ishige okamurae was found to be a potent inhibitor of bacterial PLA2 and in vivo inflammation (10). Fatty acid derivatives of bee venom and sea weeds in micro molar concentrations caused >90% inhibition of PLA2 (11). Fatty acid tricarbonyl derivatives were found to be inhibitors of human non-pancreatic cPLA2α (12). Thielocin B3 is a potent naturally occurring inhibitor of human nonpancreatic sPLA2 (13). Indole moiety constitutes the most comprehensively studied class of sPLA2 inhibitors (14). Interactions between isozymes of PLA2s and their natural and synthetic inhibitors have been reported extensively (15–24). The presence of a single molecule of lauric acid located in the active site of a Lys49-PLA2 during its preparative procedure showed the affinity between them (25).

In order to see the structural basis of the anti-inflammatory property of n-hexadecanoic acid, X-ray structure of the complex of n-hexadecanoic acid (C16H32O2):PLA2 has been determined. The kinetics parameters of PLA2 inhibition by n-hexadecanoic acid have also been determined.

Experimental Methods

  1. Top of page
  2. Abstract
  3. Experimental Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

Kinetic studies of n-hexadecanoic acid with PLA2

The assay procedure

Enzyme assay was conducted to find whether n-hexadecanoic acid inhibited PLA2 or not. The assay was carried out by spectrophotometric method as per the protocol obtained from the Sigma-Aldrich. Porcine pancreatic PLA2 and n-hexadecanoic acid were purchased from Fluka and Sigma (Bengaluru, India) respectively. Soya-lecithin of 0.015, 0.032, 0.047, and 0.063 m molar concentrations, dissolved in 10 mm calcium chloride solution in de-ionized water, were used as substrate. PLA2 solution of 1 mg/mL was prepared in de-ionized water. A total of 50 μL aliquots of enzyme solution, mixed with equal volume of 10 mm calcium chloride solution, were added to 100 μL aliquots of the substrate solutions mentioned above and incubated for 5 min at 37 °C. Fatty acid was produced from lecithin in the presence of PLA2. After incubation, the 0.2 mL reaction mixture was added to 1.5 mL ether-ethanol mixture for stopping the reaction. Then, 2 m hydroxyl amine (0.2 mL) and 14% w/v NaOH (0.2 mL) solutions were added simultaneously to it. The reaction mixtures were incubated at room temperature for 20 min, for the formation of hydroxamic acid derivatives. Afterwards, 0.30 mL each of FeCl3 (10%w/v) and HCl (3 N) were mixed with the incubated reaction mixtures, and the optical density (OD) was measured at 570 nm. The OD is proportional to the hydroxamic acid derivative formed.

The same procedure was repeated using enzyme:n-hexadecanoic acid complex. From the enzyme stock solution of 1 mg/mL, appropriate amount of PLA2 was pipetted out and mixed with n-hexadecanoic acid dissolved in DMSO/water to yield solutions of 1:10, 1:20, and 1:30 molar ratios of enzyme to inhibitor. A sample of PLA2 in the same concentration, saturated with n-hexadecanoic acid was also prepared and used to estimate the extent of inhibition. A total of 50 μL of enzyme:n-hexadecanoic acid complex was mixed with equal volume of 10 mm calcium chloride solution, added to substrate solutions, and assay was carried out. OD was measured and Line weaver-Burk plot was constructed (Figure 1). Michaelis-Menten constant (Km) and Maximal Velocity (Vmax) were determined from the plot.

image

Figure 1.  Lineweaver–Burk plot obtained by inhibition studies of various molar concentrations of n-hexadecanoic acid against PLA2.

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From the Km and Vmax obtained, the inhibitor constant, Ki, was calculated using the following equation (26) derived from Michaelis-Menten Equation:

Km = Km (1 + I0/Ki)

From the Ki value obtained, IC50 was calculated using the Cheng–Prusoff equation (27):

K i = IC50/1 + [S]/Km

Isothermal titration calorimetric study of n-hexadecanoic acid with PLA2

In order to obtain the binding constants of n-hexadecanoic acid to PLA2, isothermal titration calorimetric (ITC) analysis was performed in a Microcal VP-ITC. In all, 1.8 mL of 0.01 mm enzyme solution prepared in 2% aqueous DMSO with 5 mm CaCl2 was loaded to the cell of the ITC. Similarly, 0.3 mL of 0.2 mmn-hexadecanoic acid, prepared in the same solvent, was injected into the cell with a syringe. A constant 3.5 min time interval was maintained between two consecutive injections. The reference power and temperature were set as 10 μcal and 25 °C respectively. The stirrer speed was maintained at 309 rpm. Finally, the data obtained were fitted by non-linear least squares minimization method of the ORIGIN from Microcal. The ΔG, ΔS, ΔH, and binding constant (K) were calculated (Figure 2).

image

Figure 2.  Isothermal titration calorimetric analysis of PLA2 with n-hexadecanoic acid. The curve represents the non-linear least-squares fit of the energy released as a function of the n-hexadecanoic acid added during the titration. Raw thermal power signal (top) and plot of integrated heat versus ligand/protein molar ratio (bottom).

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Co-crystallization studies

Crystals were grown by hanging drop vapour diffusion method. PLA2 and n-hexadecanoic acid were dissolved in 0.05 m Tris-Maleate buffer, pH 7.2, and DMSO (50:50) at a molar ratio of 1:1. The concentration of PLA2 was 60 mg/mL. A total of 3 μL of protein solution was equilibrated against the reservoir solution containing 18% 2-Methyl 2, 4-pentanediol (MPD) in the same buffer. Crystals were obtained in 2 weeks.

Data collection

The X-ray diffraction data were collected on a MAR RESEARCH image plate, model No. 345, connected to a Rigaku RU 300 X-ray generator with graphite monochromator, at the Department of Crystallography and Biophysics, University of Madras. The wave length of X-rays used was 1.54 Å, and data were collected at 100 K. A total of 180 frames were recorded, each with 1 min exposure to X-rays. The image plate to crystal distance was set at 150 mm. The data were processed with iMosflm of CCP4 package to give a set with 91.1% completeness. The space group of the binary complex crystals was P3121. The unit cell parameters were a = b = 69.2 and c = 69.6 A. The data collection statistics are given in Table 1.

Table 1.   X-ray Data collection statistics of PLA2:n-hexadecanoic acid complex crystal
Data collection statistics
Space groupP3121
Cell dimensions
 a69.2 Å
 b69.2 Å
 c69.6 Å
Solvent content64.2%
Temperature100 K
Wavelength2.5 Å
No. of reflections58 568
Overall completeness91.1%
Resolution range24.55–2.50 Å

Structure solution and refinement

Molecular replacement method (MOLREP) (28) was used to solve the crystal structure. The coordinates of ppPLA2 (PDB ID: 3L30) (29) were used as the model for molecular replacement. Rigid-body refinement with maximum likelihood restraints, followed by refinement of atomic positions and isotropic temperature factors were performed with the program REFMAC 5 (30). The R and Rfree at this stage were 0.23 and 0.29 respectively. The model was fitted into the electron density map using COOT program (31). Fifty-eight water molecules and two calcium ions were identified from the difference Fourier (Fo−Fc) map. Subsequent refinement brought down the R and Rfree to 0.21 and 0.25 respectively. At this stage, a characteristic electron density in the difference Fourier map at 2.5σ was observed at the active site of PLA2. The ligand n-hexadecanoic acid fitted well in this electron density. Further, Translation Liberation Screw (TLS) & Restraint refinement brought down the R and Rfree to 0.19 and 0.24 respectively. Refinement statistics are given in Table 2.

Table 2.   X-ray structure refinement statistics of PLA2:n-hexadecanoic acid complex crystal
Refinement statistics
Rfactor19.3%
Rfree24.6%
No. of reflections used6044
RMS deviation-bond lengths0.019 Å
RMS deviation-bond angles1.93°
RMS deviation-dihedral angles21.195°
No. of protein atoms971
No. of water molecules58
Average B40.61 Å2
Average B of protein atoms40.47 Å2
Average B of ligand atoms49.53 Å2
Occupancy of ligand0.80

Binding energy calculation

Using score in place option of Schrodinger 9.1, the position of the n-hexadecanoic acid was simulated as in the crystal structure and glide score was noted. From the simulated pose, the binding free energy was calculated using MM-GBSA (32) module of the program Schrodinger 9.1.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Experimental Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

Enzyme kinetics studies revealed that the Km and Vmax of PLA2 to be 0.002 m and 200 μmol/mg/min respectively. The complex of enzyme:n-hexadecanoic acid in 1:30 molar ratio showed no change in Vmax and showed increase of Km. The Km rose to 0.008 m at the saturation level of n-hexadecanoic acid, and the Vmax remained unchanged. So, the type of inhibition was competitive (33). Usually, for raising binary complex crystals, the small ligands may be prepared with high molar concentration in solution than the enzymes. We got binary complex crystals of PLA2 and n-hexadecanoic in 10:8 molar ratio from their solution in 1:1 molar ratio (Table 2). It showed high affinity between them. n-hexadecanoic acid occupied the active site of the PLA2, preventing the binding of substrate. The inhibitor constant (Ki) was calculated to be 1.58 × 10−5 m and IC50 value was 43.26 × 10−5 m. The Lineweaver-Burk plot is given as Figure 1.

The parameter Ki, deduced from the enzyme kinetics data is related to the binding of the inhibitor to PLA2 in the presence of the substrate. The binding energy of n-hexadecanoic acid was deduced from the ITC results. The data from 29 injections were fitted using non-linear least squares fitting method at stoichiometry, n equal to 1. The binding constant (K), change in enthalpy (ΔH), and change in entropy (ΔS) were obtained from the graph, as 2.32 × 106/m, −700.9 cal/mol, and 26.8 cal/mol respectively. The binding energy (ΔG) was found to be −8.69 kcal/mol. The positive entropy (ΔS = 26.8 cal/mol/deg) indicated an entropy driven interaction between PLA2 and n-hexadecanoic acid. The binding isotherm and non-linear least-squares analysis of the data are shown in Figure 2. The top half of the Figure 2 shows the heat effect associated with successive injections until saturation is achieved and its bottom half shows the integrated heat effect associated with each injection. Usually, it is assumed that in the entropy driven interactions, the chance of formation of hydrogen bonds is very low. It is found in the present X-ray structure that the binding of n-hexadecanoic acid is stabilized by hydrophobic interactions and the hydrogen bonds are formed only outside the substrate binding cavity of the active site. Hence, the binding of n-hexadecanoic acid at the active site of PLA2 is in accordance with the above assumption on entropy driven interactions. The asymmetric unit of the crystal contained one molecule of PLA2, one molecule of n-hexadecanoic acid, two calcium ions, and 58 water molecules. The R and Rfree for the final structure were 0.19 and 0.24 respectively. The Root-Mean-Square (RMS) deviations for bond lengths and angles were 0.019 Å and 1.93°. Ramachandran Map was made to check the conformational quality of the structure. A total of 93.4% residues of the protein were in the most preferred region and 5.74% of residues in the additionally allowed regions. Average B value for all atoms was 40.61 A2 and the ligand was of average B 48.35 A2 with 0.80 occupancy. The atomic coordinates have been deposited in the Protein Data Bank (PDB ID: 3QLM). The position of the hydrophobic part of n-hexadecanoic acid in the crystal structure was inside the hydrophobic channel of active site, and the carboxylic part was towards the solvent. This orientation stabilized the position of the ligand strengthened by a water mediated, bifurcated hydrogen bond. The water 185 was found to bond with ND2 atom of ASN 67 and O2 and O atom of n-hexadecanoic acid (Figure 3). Presumably, the presence of n-hexadecanoic acid in the hydrophobic channel of the enzyme prevented the entry of the substrate and made the catalytic residues HIS 48, ASP 49, and a catalytic Ca2+ unavailable to the substrate. It was also observed that the positioning of n-hexadecanoic acid in the active site resembled the roller coaster rail. It corroborates the earlier reports (5–11). Modulation of PLA2 activity is a very important pharmacological goal for certain chronic inflammatory conditions such as rheumatoid arthritis and asthma. Extremely high levels of PLA2 were observed in synovial fluid of arthritic patients at the time of inflammation. This suggested that PLA2 may be involved in the process of inflammation and is a target for the design of anti-inflammatory drugs (20). A better understanding of how fatty acids modulate the function of cells involved might help developing of drugs based on the structure of natural inhibitors and their interaction with the target molecule. A recent review (15) of Phospholipase A2 enzymes reports the limitations of developing anti-inflammatory drugs by molecular modeling techniques combined with chemical modification of lead compounds. Only a few compounds, similar to substrate, have been found within the active site of PLA2 giving the structural details of their interaction. They were the products formed only through purification and crystallization processes (22,24) and without enzyme inhibition data (22–24). Hence, such data tend to be deficient for molecular designing.

image

Figure 3. n-hexadecanoic acid bound in the active site of PLA2. (A) Electron density of the ligand and surrounding protein residues. The hydrogen bond between PLA2 and n-hexadecanoic acid is given as broken lines. (B) n-hexadecanoic acid bound at the hydrophobic cavity shown in surface model.

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The in silico studies suggest that n-hexadecanoic acid possesses a binding energy of −58.14 Kcal/mol. It is comparable with the binding free energies of other reported inhibitors (34). So from the analysis it was concluded that binding of n-hexadecanoic acid is stable in the active site of PLA2.

Conclusion

  1. Top of page
  2. Abstract
  3. Experimental Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

It is suggested that the n-hexadecanoic acid might function as an anti-inflammatory agent, as it had shown significant inhibitory activity in the enzyme kinetics study of PLA2, entropy driven strong binding to the enzyme shown by ITC analysis, high active site binding affinity shown by forming binary complex crystals with PLA2 in their 1:1 molar solution, and its binding at the active site of the enzyme in the X-ray structure. An inhibitor appropriate only to the active site of an enzyme may cross react with several other enzymes (35). Several fatty acid derivatives implicated in inflammation management are inhibitors of PLA2 (36–38). It may be better to have substrate analogy for enzyme inhibitor models. The above analysis highlights the need of more data for structure based modeling/designing of PLA2 inhibitors. The present report may be the first to feature a naturally occurring fatty acid of human consumption and to demonstrate as a competitive inhibitor of PLA2.

Hence, a drug delivery medium, a fatty acid, itself being inhibitor of PLA2 is significant in the context of anti-inflammatory drugs meant for topical applications. The inferences from the present study indirectly validate the rigorous use of medicated oils rich in n-hexadecanoic acid for the treatment of rheumatic symptoms in the traditional medical system of India, Ayurveda. It is also significant that a drug delivery medium, fatty acid, itself being inhibitor to PLA2 in the context of anti-inflammatory drugs and their delivery.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Experimental Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References

VA thanks UGC BSR Fellowship. DKV thanks ICMR for the Senior Research Fellowship. PKM thanks CSIR for the Senior Research Fellowship. The authors gratefully acknowledge ‘G. N. Ramachandran protein crystal data collection facility’, CAS in Crystallography & Biophysics, University of Madras for the X-ray diffraction data collection facility and ‘Bioinformatics Infrastructure Facility’ (supported by DBT, Govt. of India) located at the department of Biotechnology and Microbiology, Kannur University for the computational work.

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  2. Abstract
  3. Experimental Methods
  4. Results and Discussion
  5. Conclusion
  6. Acknowledgments
  7. References
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