Water-soluble Enzyme Research Articles

Preparation and Catalytic Properties of Carbonic Anhydrase Conjugated to Liposomes through a Bis-Aryl Hydrazone Bond.

Liposomes (lipid vesicles) with sizes of about 100-200 nm carrying surface-bound (immobilized) water-soluble enzymes are functionalized molecular compartment systems for possible applications, for example, as therapeutic materials or as catalytic reaction units for running reactions in aqueous media in vitro. One way of covalently attaching enzyme molecules under mild conditions in a controlled way to the surface of preformed liposomes is to apply the spectrophotometrically traceable bis-aryl hydrazone (BAH) bond between the liposome and the enzyme molecules of interest. Using bovine carbonic anhydrase (BCA), an aqueous dispersion of liposome-BAH-BCA - conjugates of defined composition was prepared. The liposomes used consisted of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), N-(methylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), and N-(aminopropylpolyoxyethylene oxycarbonyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG-NH2). The amino group of some of the DSPE-PEG-NH2 molecules present in the liposomes were converted into an aromatic aldehyde, which (after purification) reacted with (purified) BCA molecules that had on their surface on average one acetone protected aromatic hydrazine. After purification of the liposome-BAH-BCA conjugate dispersion obtained, it was characterized in terms of (i) BCA activity, (ii) overall BCA structure, and (iii) storage stability. For an average liposome of 138 nm diameter, about 1200 BCA molecules were attached to the outer liposome surface. Liposomally bound BCA was found to exhibit (i) similar catalytic activity at 25 °C and (ii) similar storage stability when stored in a dispersed state in aqueous solution at 4 °C as free BCA. Measurements at 5 °C clearly showed that liposome-BAH-BCA is able to catalyze the hydration of carbon dioxide to hydrogen carbonate.

Where Does an Enzyme Reside in a Bicontinuous Structure?

Using enzymes as catalysts in biochemical processes requires to bring them into close contact with the substrates to be processed. For oil-soluble substrates and water soluble enzymes this might be achieved by bringing the water and oil phase in a bicontinuous microemulsion into close contact. In this contribution we review two possible scenarios of how the enzyme and the interface influence each other. Small angle neutron scattering (SANS) and neutron spin echo spectroscopy (NSE) act as a microscope to look into the details of the interfacial region of microemulsions.

A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Screening Lipolytic from Soil Bacterial Contaminated Oil

Lipase (triasilglycerol acylhydrolase, E.C, 3.1.1.3) is an enzyme can hydrolyze long chains of triglycerides[1]. Lipase is a water-soluble enzyme and naturally lipase catalyze the hydrolysis of ester bonds in water insoluble lipid substrate and long-chain acylglycerol synthesis, diacylglycerol, monoacylglycerol, and glycerol[2]. Hydrolase enzyme lipase can be used potentialy produced fatty acid which precursors in the chemical industry[3]. The research aims to study the lipolytic activity of bacteria isolated from soil contaminated with oil. Screening of lipolytic bacteria was performed in Rhodamin-B agar plate containing olive oil. The lipolytic activity was determined by spectrofotometry method toward p-nitrofenil palmitat as a substrate. The result this research showed that there are 15 of 150 isolates bacteria from soil contaminated with oil was confirmed by observing the zone of hydrolysis formed around the growth of colony on Rhodamin B agar plate containing olive oil. One of the lipolytic bacteria identified as higher activity was produced lipase with activity of 7,41 U/ml

Lipases: it's not just pancreatic lipase!

Lipases are water-soluble enzymes that hydrolyze water-insoluble lipid molecules, such as triglycerides, phospholipids, and galactolipids. They are ubiquitous in nature and are present in humans, animals, insects, plants, fungi, and microorganisms. While we commonly consider pancreatic lipase, this review provides an overview of several lipases that are important for the digestion and metabolism of lipids in veterinary species. All of these enzymes have specific functions but share a common α/β-hydrolase fold and a catalytic triad where substrate hydrolysis occurs. The pancreatic lipase gene family is one of the best characterized lipase gene families and consists of 7 mammalian subfamilies: pancreatic lipase, pancreatic lipase related proteins 1 and 2, hepatic lipase, lipoprotein lipase, endothelial lipase, and phosphatidylserine phospholipase A1. Other mammalian lipases that play integral roles in lipid digestion include carboxyl ester lipase and gastric lipase. Although most enzymes have preferred substrate specificity, much overlap occurs across the plethora of lipases because of the similarities in their structures. This has major implications for the development and clinical utilization of diagnostic assays. These implications are further explored in our companion Currents in One Health article by Lim et al in the August 2022 issue of the Journal of American Veterinary Medical Association, which focuses on pancreatic lipase assays for the diagnosis of pancreatitis.

Exploring the Pivotal Neurophysiologic and Therapeutic Potentials of Vitamin C in Glioma

Gliomas represent solely primary brain cancers of glial cell or neuroepithelial origin. Gliomas are still the most lethal human cancers despite modern innovations in both diagnostic techniques as well as therapeutic regimes. Gliomas have the lowest overall survival rate compared to other cancers 5 years after definitive diagnosis. The dietary intake of vitamin C has protective effect on glioma risk. Vitamin C is an essential compound that plays a vital role in the regulation of lysyl and prolyl hydroxylase activity. Neurons store high levels of vitamin C via sodium dependent-vitamin C transporters (SVCTs) to protect them from oxidative ischemia-reperfusion injury. Vitamin C is a water-soluble enzyme, typically seen as a powerful antioxidant in plants as well as animals. The key function of vitamin C is the inhibition of redox imbalance from reactive oxygen species produced via the stimulation of glutamate receptors. Gliomas absorb vitamin C primarily via its oxidized dehydroascorbate form by means of GLUT 1, 3, and 4 and its reduced form, ascorbate, by SVCT2. Vitamin C is able to preserve prosthetic metal ions like Fe2+ and Cu+ in their reduced forms in several enzymatic reactions as well as scavenge free radicals in order to safeguard tissues from oxidative damage. Therapeutic concentrations of vitamin C are able to trigger H2O2 generation in glioma. High-dose combination of vitamin C and radiation has a much more profound cytotoxic effect on primary glioblastoma multiforme cells compared to normal astrocytes. Control trials are needed to validate the use of vitamin C and standardization of the doses of vitamin C in the treatment of patients with glioma.

The activity of the intrinsically water-soluble enzyme ADAMTS13 correlates with the membrane state when bound to a phospholipid bilayer

Membrane-associated enzymes have been found to behave differently qualitatively and quantitatively in terms of activity. These findings were highly debated in the 1970s and many general correlations and reaction specific models have been proposed, reviewed, and discarded. However, new biological applications brought up the need for clarification and elucidation. To address literature shortcomings, we chose the intrinsically water-soluble enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) and large unilamellar vesicles with a relative broad phase transition. We here present activity measurements of ADAMTS13 in the freely dissolved state and the membrane associated state for phosphocholine lipids with different acyl-chain lengths (13:0, 14:0 and 15:0) and thus main phase transition temperatures. While the freely dissolved enzyme shows a simple Arrhenius behavior, the activity of membrane associated ADAMTS13 in addition shows a peak. This peak temperature correlates with the main phase transition temperature of the used lipids. These findings support an alternative theory of catalysis. This theory predicts a correlation of the membrane associated activity and the heat capacity, as both are susceptibilities of the same surface Gibb’s free energy, since the enzyme is attached to the membrane.

Abstract P112: A Simple, Rapid, And Sensitive Fluorescence-based Method To Assess Triacylglycerol Hydrolase Activities

Introduction: Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their activity is usually measured after isolation and quantification of the hydrolyzed free fatty acids. We have used NBD-labeled lipids previously to develop TAG, cholesteryl ester, and phospholipid transfer assays for microsomal triglyceride transfer protein. To date, however, NBD-labeled TAG (NBD-TAG) has not been used to assess the enzymatic activities of different lipases. Methods: We developed methods to measure lipase activities using adipose triglyceride lipase (ATGL), lipoprotein lipase (LpL), and pancreatic triglyceride lipase (PNLIP) as model lipases. In these assays, we incubated a source of ATGL, LpL, or PNLIP with substrate emulsions containing nitrobenzoxadiazole (NBD)-labeled TAG in phosphatidylcholine (PC) and phosphatidylinositol (PI), measured increases in NBD fluorescence with time, and calculated enzyme activities. Results: Incorporation of NBD-TAG into PC vesicles resulted in some hydrolysis; but required product isolation step to measure NBD fluorescence. However, incorporation of PI into these NBD-TAG/PC vesicles significantly increased substrate hydrolysis in time, protein, and substrate concentration dependent manner negating the need to isolate products to measure lipase activities. Further, increasing the ratio of NBD-TAG to PC enhanced substrate hydrolysis. Next, we tested specific lipase inhibitors and found that orlistat inhibits all three enzymes indicating that it is a pan-lipase inhibitor. Conclusions: We describe a simple, rapid fluorescence-based TAG hydrolysis assay to assess three major TAG hydrolases: ATGL localized intracellularly, LpL localized at the extracellular endothelium, and PNLIP produced and secreted from the pancreas into the intestinal lumen. The major advantages of this method are its speed, simplicity, and avoidance of a product isolation step. This assay is potentially applicable to a wide range of lipases and is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and in the diagnosis of diseases associated with increases in plasma lipase activities.

Reconstitution of GABA, Glycine and Glutamate Transporters

In contrast to water soluble enzymes which can be purified and studied while in solution, studies of solute carrier (transporter) proteins require both that the protein of interest is situated in a phospholipid membrane and that this membrane forms a closed compartment. An additional challenge to the study of transporter proteins has been that the transport depends on the transmembrane electrochemical gradients. Baruch I. Kanner understood this early on and first developed techniques for studying plasma membrane vesicles. This advanced the field in that the experimenter could control the electrochemical gradients. Kanner, however, did not stop there, but started to solubilize the membranes so that the transporter proteins were taken out of their natural environment. In order to study them, Kanner then had to find a way to reconstitute them (reinsert them into phospholipid membranes). The scope of the present review is both to describe the reconstitution method in full detail as that has never been done, and also to reveal the scientific impact that this method has had. Kanner’s later work is not reviewed here although that also deserves a review because it too has had a huge impact.

A simple, rapid, and sensitive fluorescence-based method to assess triacylglycerol hydrolase activity

Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluorescence method to measure lipase activity in real time that does not require the separation of substrates from products. We developed this method using adipose triglyceride lipase (ATGL) and lipoprotein lipase (LpL) as model lipases. We first incubated a source of ATGL or LpL with substrate vesicles containing nitrobenzoxadiazole (NBD)-labeled TAG, then measured increases in NBD fluorescence, and calculated enzyme activities. Incorporation of NBD-TAG into phosphatidylcholine (PC) vesicles resulted in some hydrolysis; however, incorporation of phosphatidylinositol into these NBD-TAG/PC vesicles and increasing the ratio of NBD-TAG to PC greatly enhanced substrate hydrolysis. This assay was also useful in measuring the activity of pancreatic lipase and hormone-sensitive lipase. Next, we tested several small-molecule lipase inhibitors and found that orlistat inhibits all lipases, indicating that it is a pan-lipase inhibitor. In short, we describe a simple, rapid, fluorescence-based triacylglycerol hydrolysis assay to assess four major TAG hydrolases: intracellular ATGL and hormone-sensitive lipase, LpL localized at the extracellular endothelium, and pancreatic lipase present in the intestinal lumen. The major advantages of this method are its speed, simplicity, and elimination of product isolation. This assay is potentially applicable to a wide range of lipases, is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and can be used for diagnostic purposes.

Morpho-physiological and biochemical changes in finger millet [Eleusine coracana (L.) Gaertn.] underdrought stress.

Finger millet (Eleusine coracana L. Gaertn.) is a nutritious and climate-resilient crop with a C4 type carbon fixation pathway. The present study was aimed to assess the drought tolerance capacities of four finger millet genotypes based on their physiological and biochemical characteristics at three different phenological stages. Finger millet genotypes RAU 8, GPU 67, GPU 28 and MS 9272 were subjected to two water regimes, regular irrigation (control) and suspended irrigation (drought stress). During water regimes, morpho-physiological [biomass accumulation, leaf relative water content, and photosynthetic pigments] and biochemical changes [proline content, water soluble carbohydrates, antioxidant enzymes, and malondialdehyde content] were studied during seedling (18th day), vegetative (49th day) and early flowering stages (73rd day). The maintenance of growth especially root growth, biomass accumulation, the differential response in the concentration and changes of pigments, accumulation of proline, water-soluble carbohydrates and increased levels of antioxidant enzymes under drought stress play a major role in differential tolerance in finger millet genotypes that is conferred by the biplot analysis. The genotype RAU 8 is the most drought-tolerant genotype at all the three different phenological stages. Whereas the genotype GPU 67 was identified as sensitive at the seedling stage and its tolerance level was improved at vegetative and early flowering stages. The genotypes GPU 28 and MS 9272 were considered as drought sensitive at all three different phenological stages. Our results provide inputs to the breeders to select genotypes as parents and to design effective strategies in crop improvement programs.

Modulation of non-bilayer lipid phases and the structure and functions of thylakoid membranes: effects on the water-soluble enzyme violaxanthin de-epoxidase

The role of non-bilayer lipids and non-lamellar lipid phases in biological membranes is an enigmatic problem of membrane biology. Non-bilayer lipids are present in large amounts in all membranes; in energy-converting membranes they constitute about half of their total lipid content—yet their functional state is a bilayer. In vitro experiments revealed that the functioning of the water-soluble violaxanthin de-epoxidase (VDE) enzyme of plant thylakoids requires the presence of a non-bilayer lipid phase. 31P-NMR spectroscopy has provided evidence on lipid polymorphism in functional thylakoid membranes. Here we reveal reversible pH- and temperature-dependent changes of the lipid-phase behaviour, particularly the flexibility of isotropic non-lamellar phases, of isolated spinach thylakoids. These reorganizations are accompanied by changes in the permeability and thermodynamic parameters of the membranes and appear to control the activity of VDE and the photoprotective mechanism of non-photochemical quenching of chlorophyll-a fluorescence. The data demonstrate, for the first time in native membranes, the modulation of the activity of a water-soluble enzyme by a non-bilayer lipid phase.

Facile strategy for immobilizing horseradish peroxidase on a novel acetate functionalized ionic liquid/MWCNT matrix for electrochemical biosensing

Facile yet simple platforms for the immobilization of biomolecules have always been a substantial requirement for the fabrication of proficient biosensors. In this study, we report a naphthyl substituted acetate functionalized ionic liquid (NpAc-IL) for the covalent anchoring of horseradish peroxidase (HRP), using which the direct electrochemistry of HRP was successfully accomplished and a H2O2 biosensor was developed. The naphthyl substitution on the NpAc-IL was utilized for the π-π stacking with the MWCNT modified GCE and the terminal -OCH3 group of NpAc-IL was used for the covalent attachment with the free -NH2 group of HRP via amide bond formation. High conducting nature of the newly designed ionic liquid (NpAc-IL), facilitated an improved communication with the deeply buried redox centre of the HRP, while the covalent bonding provided enhanced stability to the fabricated biosensor by stably holding the water soluble HRP enzyme on the electrode surface. Furthermore, incorporation of MWCNT on the sensor setup synergistically enhanced the sensitivity of the developed biosensor. Under optimized conditions, the fabricated biosensor showed an enhanced electrocatalytic reduction of H2O2 in the range of 0.01 to 2.07 mM with a limit of detection and sensitivity of 2.7 μM and 55.98 μA mM−1 cm−2 respectively. Further, the proposed biosensor was utilized for the sensing of H2O2 spiked in real samples. Moreover, the newly fabricated biosensor demonstrated excellent stability with improved sensitivity and selectivity towards H2O2 reduction. The superior analytical characteristics are attributed to the facile fabrication strategy using this newly developed acetate functionalized ionic liquid platform.

Bioinspired Dual-Enzyme Colloidosome Reactors for High-Performance Biphasic Catalysis.

In this paper, a novel method for the construction of colloidosomes as a microreactor for dual-enzyme cascade biphasic reaction has been reported. A lipase-glucose oxidase (GOx) enzyme pair is employed in this system. A water-soluble enzyme GOx is compartmentalized inside the colloidosomes. A hydrophobic environment-favored enzyme Candida Antarctica lipase B (CalB) is adsorbed on the outer surfaces of the colloidosomes. The catalysis system is set up by introducing these dual-enzyme-immobilized microcapsules into acetic ether. H2O2 is produced in the aqueous phase by the doped GOx, and then H2O2 diffused out of the microcapsules is utilized by CalB to catalyze the oxidation of ethyl acetate. Finally, the formed peracids oxidized N-heteroaromatic in situ. Furthermore, no obvious yield decline is observed in four reaction cycles. Thus, our work provides a new strategy for the design of high-performance biomimicking reactors for multiple enzyme cascade reactions and further expands the potential application area of colloidosomes.

Molecular mechanism of activation of Burkholderia cepacia lipase at aqueous-organic interfaces.

Lipases are water-soluble enzymes that catalyze the hydrolysis of lipids. Since lipids are mostly hydrophobic, lipase activity occurs preferentially at interfaces of aqueous and organic phases. In this work, we study the molecular mechanisms by which the Burkholderia cepacia lipase (BCL) is activated at interfaces of water with octane and with methyl caprylate (CAME). We show that BCL assumes very rapidly a preferential orientation at the interfaces, in which the active site is exposed to the organic phase. With BCL oriented to the interface, we compute the free energy of the aperture of the catalytic pocket using Adaptive Biasing Force MD simulations. The exposure to the organic phase promotes a clear stabilization of the open form of the catalytic pocket relative to the enzyme in water. This stabilization stems from the hydrophobicity of domains U1 and U2, which allows the penetration of organic solvents into the catalytic cleft impeding the closure of the pocket. Our results suggest that the structure and hydrophobicity of BCL are optimized for its activation in biphasic systems through the regulation of the accessibility of the catalytic pocket by, and for, hydrophobic substrates. The understanding of this mechanism may be useful for the design of proteins with targeted activation.

Physicochemical properties of quinoa flour as affected by starch interactions

There has been growing interest in whole grain quinoa flour for new product development due to the unique nutritional benefits. The quality of quinoa flour is much determined by the properties of its major component starch as well as non-starch components. In this study, composition and physicochemical properties of whole grain flour from 7 quinoa samples have been analyzed. Flour properties have been correlated to the flour composition and the properties of isolated quinoa starches through chemometrics. Great variations in chemical composition, swelling power, water soluble index, enzyme susceptibility, pasting, gel texture, and thermal properties of the flour have been observed. Correlation analysis showed that thermal properties and enzyme susceptibility of quinoa flour are highly influenced by the starch. Interactions of starch with non-starch components, including lipids, protein, dietary fibre, phenolics, and minerals, greatly impacted the flour properties. For example, peak gelatinization temperature of the flour is positively correlated to that of the starch (r=0.948, p<0.01) and negatively correlated to the lipid content (r=-0.951, p<0.01). Understanding the roles of starch and other components in physicochemical properties of quinoa flour provides a basis for better utilization of this specialty crop.

High Internal Phase Emulsion Ring‐Opening Polymerization of Pentadecanolide: Strategy to Obtain Porous Scaffolds in a Single Step

Single‐step process for production of porous scaffold based on poly(pentadecanolide) (PPDL) is reported. The scaffold is produced via in situ porosity generation during ring‐opening polymerization (ROP) of the lactone pentadecanolide (PDL) in high internal phase emulsion (HIPE) using a water soluble enzyme (Lipase TL) as catalyst. The enzyme present in dispersed aqueous phase of HIPE effectively carries out ROP of monomer present in continuous oil phase. The polymerization occurs at the interface of oil and water in HIPE and complete monomer conversion is achieved in 96 h at 40 °C. Porous scaffold having interconnected pores is obtained as the final product and formation of PPDL is confirmed by spectroscopic and thermal analysis. The process thus developed is a single‐step process to produce porous scaffolds via ROP of a lactone that can be extended to see its efficacy using monomer‐soluble organometallic catalysts and monomers other than PDL including lactones, lactides, and cyclic carbonates. image

Thermoreversible and Injectable ABC Polypeptoid Hydrogels: Controlling the Hydrogel Properties through Molecular Design.

A series of ABC triblock copolypeptoids [i.e., poly(N-allyl glycine)-b-poly(N-methyl glycine)-b-poly(N-decyl glycine) (AMD)] with well-defined structure and varying composition have been synthesized by sequential primary amine-initiated ring-opening polymerization of the corresponding N-substituted N-carboxyanhydride monomers (Al-NCA, Me-NCA, and De-NCA). The ABC block copolypeptoids undergo sol-to-gel transitions with increasing temperature in water and biological media at low concentrations (2.5-10 wt %). The sol-gel transition is rapid and fully reversible with a narrow transition window, evidenced by the rheological measurements. The gelation temperature (Tgel) and mechanical stiffness of the hydrogels are highly tunable: Tgel in the 26.2-60.0 °C range, the storage modulus (G') and Young's modulus (E) in the 0.2-780 Pa and 0.5-2346 Pa range, respectively, at the physiological temperature (37 °C) can be readily accessed by controlling the block copolypeptoid composition and the polymer solution concentration. The hydrogel is injectable through a 24 gauge syringe needle and maintains their shape upon in contact with surfaces or water baths that are kept above the sol-gel transition temperature. The hydrogels exhibit minimal cytotoxicity toward human adipose derived stem cells (hASCs), evidenced from both alamarBlue and PicoGreen assays. Furthermore, quantitative PCR analysis revealed significant up-regulation of the Col2a1 gene and down-regulation of ANGPT1 gene, suggesting that the hydrogel exhibit biological activity in inducing chondrogenesis of hASCs. It was also demonstrated that the hydrogel can be used to quantitatively encapsulate water-soluble enzymes (e.g., horseradish peroxidase) by manipulating the sol-gel transition. The enzymatic activity of HRP remain unperturbed after encapsulation at 37 °C for up to 7 d, suggesting that the hydrogel does not adversely affect the enzyme structure and thereby the enzymatic activity. These results suggest that the polypeptoid hydrogel a promising synthetic platform for tissue engineering or protein storage applications.

Influence of small-molecule ligands and their complexes on lysozyme properties

The structure of complexes of various alkylhydroxybenzenes, such as 4-hexylresorcinol, 5-methyl-resorcinol, and tyrosol, is studied using experimental and theoretical methods. The influence of 4-hexyl-resorcinol and 5-methylresorcinol in a wide range of concentrations on the structure, equilibrium fluctuations, and functional activity of a water-soluble enzyme lysozyme is examined. A spatial model for the interaction of ligands and clusters thereof with the protein and the aqueous medium is constructed. A possible mechanism of the stabilization of the protein tertiary structure by the aforementioned molecules is proposed.

Modeling and Inhibitor Design of Ca(2+)-Independent Phospholipase A2

Ca(2+)-independent phospholipase A2 (iPLA2) is a water-soluble enzyme that is active in association with lipid membranes. The deregulation of iPLA2 causes uncontrolled inflammation that plays a role in a large number of diseases. The design of selective inhibitors of iPLA2 has been complicated by the absence of any X-ray or NMR information about the enzyme structure. Here we have carried out microsecond MD simulations on homology models of iPLA2 to reach a complete understanding of the dynamic and membrane-related processes associated with the enzyme function and inhibition. The simulations help answer questions that have challenged scientists in this field for decades about the nature of the protein-bilayers complex, the importance of dynamics in the regulation of the active-site pocket, and the detailed mechanism of lipid extraction.View Large Image | View Hi-Res Image | Download PowerPoint Slide