Sandbox 51

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= '''Lysozyme''' =
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<applet load='1hpl' size='400' frame='true' align='right' caption='Lipase' />
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==Introduction==
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<scene name='Sandbox_51/1b67_salt_bridges/2'>hmfa2 salt bridges</scene>
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= Introduction =
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<scene name='Sandbox_51/H_bonds/2'>diff residues</scene>
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Lysozyme - also known as muramidase, or glycoside hydrolase - is a powerful enzyme of biological significance found in abundance in tears, saliva, and human milk. In humans, it is encoded in the ''LYZ'' gene. Although it is responsible for the initial digestion of starches in the mouth, it is most widely identified as a non-specific defense in gram positive bacteria and in many species of fungi. Due to its antibacterial effects, it is a strong component of the innate immune system, and is an important part of an infant's diet to ward off diarrheal diseases. Since it is a small, easily available, and highly stable protein containing only 129 amino acid residues, it has been subject to extensive research regarding its function and structure. Hen Egg White (HEW) Lysozyme is shown below.
 
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'''History'''
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<scene name='Sandbox_51/Hmfa2/1'>TextToBeDisplayed</scene>
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Lysozyme is an enzyme known for its unique ability to degrade the polysaccharide architecture of many kinds of cell walls, normally for the purpose of protection against bacterial infection<ref>Lysozyme. 2010. Citizendium.org. http://en.citizendium.org/wiki/Lysozyme</ref>. Its effects were first noticed by Laschtschenko in 1909. It was officially characterized and termed “lysozyme” by Alexander Fleming, the same person credited for the accidental discovery of penicillin.
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<scene name='Sandbox_51/1b67_salt_bridges/3'>good</scene>
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The characterization of lysozyme in 1922 by Alexander Fleming was providential in that the undertaken experiment related to the discovery of lysozyme was not geared toward any knowledge of such a protein as lysozyme <ref>Lysozyme. 2008. Lysozyme.co.uk. http://lysozyme.co.uk/</ref>. During the unrelated experiment, nasal drippings were inadvertently introduced to a petri dish containing a bacterial culture, which culture consequently exhibited the results of an as yet unknown enzymatic reaction. The observation of this unknown reaction led to further research on the components of this reaction as well as to the corresponding identification of the newfound "lysozyme." Fleming's discovery was complemented by David C. Phillips' 1965 description of the three-dimensional structure of lysozyme via a 200 pm resolution model obtained from X-ray crystallography <ref>Lysozyme, 2008. Lysozyme.co.uk. http://lysozyme.co.uk/</ref>. Phillips' work was especially groundbreaking since Phillips had managed to successfully elucidate the structure of an enzyme via X-ray crystallography - a feat that had never before been accomplished<ref>Bugg, T. 1997. An Introduction to Enzyme and Coenzyme Chemistry. Blackwell Science Ltd., Oxford </ref>. Phillips' research also led to the first sufficiently described enzymatic mechanism of catalytic action <ref>1967. Proc R Soc Lond B Bio 167 (1009): 389–401.</ref>. Thus, Phillips' elucidation of the function of lysozyme led Phillips to reach a more general conclusion on the diversity of enzymatic chemical action in relation to enzymatic structure. Clearly, the findings of Phillips as well as the more general historical development of the understanding of the structure and function of lysozyme have been paramount to the more general realm of enzyme chemistry.
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<scene name='Sandbox_51/Salt_bridges_hmfb/2'>salthmfb</scene>
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<scene name='Sandbox_51/Hmfb_hdiff_res/1'>hmfb hbonds</scene>
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[[Image:nag-nam2.jpg|thumb|left|350px|Lysozyme Cleavage Site]]
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Pancreatic lipase 1hpl (EC 3.1.1.3) is a an enzyme involved with the digestion and absorption of triacylglycerols (fats) in the intestine. It is secreted by the pancreas into the duodenum where it participates in the initial stages of breaking down fats into glyercol and three fatty acids. Lipase is a serine protease and a polar molecule. The mechanism of action requires Calcium ligands for stability and the coenzyme, colipase, for activation of the lipase mechanism. The hydrolysis products diffuse across enterocyte membrane and are absorbed into the blood <ref>Voet, D.,etc. "Fundamentals of Biochemistry: Life at the Molecular Level" John Wiley and Sons, Inc: New Jersey, 2008.</ref>. Due to that role lipase has in digestion of fat, and the growing problem of obesity, there has been increased effort to develop lipase inhibitors for weight loss supplements.
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<ref>Image from: http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/proteins/lysozyme.htm</ref>
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'''Function'''
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==Structure==
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1hpl is the horse pancreatic lipase enzyme that is thought to have a similar structure and function to the human pancreatic lipase. The <scene name='Sandbox_51/Lipase_crystal_structure/1'> Crystal Structure</scene> of the human pancreatic lipase has not yet been published. Lipase is a dimer of two <scene name='Sandbox_51/Lipase_crystal_structure/3'>monomers</scene> with 449 amino acid residues interacting with one <scene name='Sandbox_51/Ligand_position/2'>Calcium Ligand</scene> each. The calcium ion <scene name='Sandbox_51/Calcium_orientation/2'>orientation</scene> shows that the molecule is located between acidic residues Arg, Asp and Glu. The enzyme has <scene name='Sandbox_51/Nc_terminal/1'>two domains</scene> of various composition specific for certain interactions, an N-terminal (blue) and a C-terminal (red). In the <scene name='Sandbox_51/Secondary_structure/2'>secondary structure</scene>, the N-terminal domain has the hydrolase alpha/beta folding structure, consisting of an 8 stranded alpha-beta sheet connected by helices. The C-terminal domain (where enzyme colipase binds) has a beta-sheet sandwich folding pattern <ref>Horse pancreatic lipase...</ref>. The enzyme has 13 alpha helices (pink) and 22 beta sheets (yellow) per subunit, as displayed in the <scene name='Sandbox_51/Secondary_structure/2'>secondary structure</scene> <ref>Egloff, M.P., etc. "The 2.46 angstroms resolution structure of the pancreatic lipase colipase complex inhibited by a C11 alkyl phosphonate."(1995) J. Biochemistry 34: 2751-2762 [http://www.pdb.org/pdb/explore/explore.do?structureId=1lpb]</ref>. At the <scene name='Sandbox_51/Monomer_interface/1'>interface</scene> of the two monomers, interactions include 4 hydrogen bonds and 4 salt bridges that stabilize the dimer <ref>"HORSE PANCREATIC LIPASE. THE CRYSTAL STRUCTURE AT 2.3 ANGSTROMS RESOLUTION." http://bisc.cse.ucsc.edu/pages/BiscHom/1hpl_AB.html</ref>. The molecule has a varying degree of <scene name='Sandbox_51/Polar_structure/3'>polar and hydrophobic residues</scene> interspersed within the protein(polar are shown in purple and hydrophobic in white) <ref>Bourne, Y., etc. "Horse pancreatic lipase..."(1994) J.Mol.Biol. 238: 709-732 [http://www.pdb.org/pdb/explore/explore.do?structureId=1HPL]</ref>. This is important because the enzyme actively digests at the lipid-water interface of the fatty micelles, requiring stability in both polar and non polar environments <ref>Fundamentals of Biochemistry...</ref>. Within each monomer and dimer structure, the molecules are held together by disulfide bonds, hydrogen bonding, and electrostatic interactions (salt bridges). The enzyme has six covalent <scene name='Sandbox_51/Disulfide_bons/1'>disulfide bonds</scene> per monomer. Also, the <scene name='Sandbox_51/Salt_bridges/3'>salt bridges</scene> stabilize the monomers and dimer of the enzyme at positively charged nitrogens (blue) in Arg and Lys, and negative oxygens (red) in Asp and Glu residues. <scene name='Sandbox_51/Hydrogen_bonds/2'>Hydrogen bonds</scene> are present within each monomer, as shown by the hydrogen bond forming residues (light gray), and the oxygen (red) and nitrogen (blue) atoms involved in the hydrogen bonding. Lipase is water soluble due to the polar residues on the surface, but has hydrophobic sequences on the interior. At enzyme activation and interaction with colipase, a confirmation change occurs to expose the more hydrophobic regions to the nonpolar lipid micelle.
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Lysozyme is known for damaging bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid (NAM) and N-acetyl-D-glucosamine (NAG) residues in peptidoglycan, and between N-acetyl-D-glucosamine residues in chitodextrins. In this way, lysozyme is efficient in lysing the cell walls of both bacteria and fungi. The location of cleavage for lysozyme on this architectural theme is the β(1-4) glycosidic linkage connecting the C1 carbon of NAM to the C4 carbon of NAG.
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==Active Site==
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The <scene name='Sandbox_51/Active_site/3'>active site</scene> of the lipase molecule is found in the N-terminal domain (residues 1-336) and contains a <scene name='Sandbox_51/Catalytic_tria/1'>catalytic triad</scene> consisting of a Ser152-His263-Arg176 for the ester hydrolysis reaction (similar to that of a serine protease). The catalytic triad and regions around it are thought to be the best conserved aspects of lipase throughout the lipase family. The active site is covered by a 25-residue helical 'lid' blocking the binding site <ref>Fundamentals of Biochemistry...</ref>. <scene name='Sandbox_51/Lipase_and_colipase/2'>Lipase and colipase</scene> in complex with a triacylglyceride shows the unique lid (yellow) and beta-5 loop (pink) essential to catalysis. The 10 residue beta-5 loop changes confirmation when colipase binds, exposing lipase's oxyanion hole and hydrophobic surface. Before colipase binding however, lipase is in the <scene name='Sandbox_51/Closed_lid/4'>closed lid</scene> structure where the beta-5 loop (residues 76-84, in pink) and lid (residues 237-261, yellow) protect the oxyanion hole from solvent interaction. The <scene name='Sandbox_51/Mutated_open_ring/6'>open ring</scene> structure is accompanied by peptide shift which increase the hydrophobic surface area initiating the reaction with the lipid. one such important shift is at residues 240-253 (in yellow), in the lid structure, very close to the active site. Also, it is evident from the image that part of the beta-5 loop interacts with colipase in the open state. The lid opening is accompanied by a change in secondary structure from a mostly beta-extended confirmation to a structure where more than half the active site is formed from alpha helices <ref> Thomas, A. etc. "Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity", The Journal of Biological Chemistry, 2005 September 22; 270 (48): 40074-40083. </ref>.
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The particular substrate of preference for this cleavage type is a (NAG-NAM)₃ hexasaccharide, within which substrate occurs the
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[[Image:F1.medium.gif]] <ref>"Role of the Lid Hydrophobicity Pattern in Pancreatic
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cleaving target glycosidic bond, NAM₄-β-O-NAG₅. The individual hexasaccharide binding units are designated A-F, with NAM₄-β-O-NAG₅ glycosidic bond cleavage preference corresponding to a D-E unit glycosidic bond cleavage preference.
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Lipase Activity"...</ref>.
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==Function==
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[[Image:Fatty_acid_breakdown.gif]]<ref>http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/lipoprot.htm </ref>
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Pancreatic lipase initiates the breakdown of triacylglycerols into 1,2-diacylglycerols and fatty acids. Lipase is essential to hydrolyzing the very nonpolar triacylglycerols into more polar compounds capable of diffusing across enterocyte membranes, and eventually into circulation. The digestion of triacylglycerols is a major source of energy storage in metabolism. Consequently, lipase hydrolyzes the ester bonds of the triglycerides. Lipase, as a digestive enzyme, is soluble in water but the triacylglycerides it hydrolyzes are not. In the duodenum, bile salts initiate lipase's mechanism by emulsification of the fatty acids. Bile salts are usually sodium with the deprontated form of digestive acids (mostly derivatives of cholic acid). Bile salts are stored by the gallbladder and released into the duodenum where they facilitate the formation of micelles. Bile salts or acids are amphipathic. Therefore, the hydrophobic region surrounds the fatty acids and the hydrophilic remains on the outside in the process of emulsification, creating smaller micelle molecules. Micelle formation is essential to maximize the efficiency of lipase by increasing the hydrophilic surface area on which lipase will work <ref>Bowen, R. Absorption of Lipids. 8 August 2007. 3 March 2012. <http://www.vivo.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.html>.</ref>. For the hydrolysis reaction, lipase activity requires bile acids, its coenzyme (colipase), and calcium ions to achieve the correct orientation to hydrolyze the fats <ref>Fundamentals of Biochemistry...</ref>.
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<applet load='1hpl' size='300' frame='true' align='right' caption='Lipase' />
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==Mechanism==
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In addition to the effects of bile salts, Lipase is activated by the coenzyme colipase, which binds to the C-terminal non-catalytic domain of lipase. Upon binding, active lipase is stabilized for the hydrophobic interaction with the triacylglycerides <ref>Fundamentals of Biochemistry...</ref>. Colipase must be present for activation of lipase and acts as a bridge between lipase and the lipid <ref>Crandall,W., Lowe, M. "Colipase Residues Glu64 and Arg65 Are Essential for Normal Lipase-mediated Fat Digestion in the Presence of Bile Salt Micelles" Journal of Biological Chemistry, 2001, (276) 12505-12512</ref>. Colipase is also secreted in the pancreas, but in its inactive form, which must be activated by trypsin before interacting with lipase. Colipase is a small protein cofactor with 5 conserved disulfide bonds <ref>"Colipase". Wikipedia: The Free Encyclopedia. 5 July 2011 [http://en.wikipedia.org/wiki/Colipase]</ref>, and 2 surfaces- a hydrophilic surface (site of lipase C-terminal interaction) and a hydrophobic surface (contains multiple hydrophobic loops to bridge the lipid)<ref>"Colipase Residues..."</ref>. Colipase and lipase <scene name='Sandbox_51/Contacts/3'>contacts</scene> are opposite of the active site on the C-terminal (contacts are regions of pink and yellow, with water molecules shown in darker blue). The enzymes are bound by polar interactions such as <scene name='Sandbox_51/Salt_bridges/2'>salt bridges</scene> and van der waals forces <ref>van Tilbeurgh H, etc."Structure of the pancreatic lipase-procolipase complex", 1992 Sep 10;359(6391):159-62. PMID:1522902.[http://www.proteopedia.org/wiki/index.php/1n8s]</ref>. Specifically, interactions at the <scene name='Sandbox_51/Colipase_lipase_interface/1'>lipase-colipase interface</scene> include bonding between residues Glu15 and Arg38 with the lipase polar lid; and Arg44, Glu45, Glu64, Arg65, and Asn89 residues with main chain, C-terminal lipase residues <ref>"Colipase Residues..."</ref>.
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Lipase activation at the lipid-water interface of triacylglycerides, in the presence of colipase and bile salts, is known as interfacial activation. For the hydroloysis reaction to take place, colipase anchors lipase to the lipid-water membrane of the micelle and a surface change occurs on lipase. Colipase's 4 hydrophobic loops interact with the hydrophobic atmosphere of the triacylglyceride initiating the lipase active site binding to the lipid, and lid opening to reveal a more hydrophobic environment for the triacylglycerol. Once colipase is bound, lipase initiates a serine-like hydrolysis involving the His-Asp-Ser active site residues in the catalytic triad <ref>Fundamentals of Biochemistry...</ref>. In the reaction, serine attacks the ester, forming an acyl-enzyme intermediate. The His and Asp residues help to stabilize the oxyanion intermediate through hydrogen bonding. Water enters the active site and reacts to release lipase and free the fatty acid. This acylation and deacylation reaction is usually completely reversible.
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Lipase Triacylglyceride Hydrolysis Mechanism
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[[Image:F6.medium.gif]] <ref> Reetz, Manfield F. Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications. PNAS: 12 April 2004 [http://www.pnas.org/content/101/16/5716.full]</ref>.
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<applet load='1hpl' size='230' frame='true' align='left' caption='Lipase' />
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= Enzymatic Activity of Lysozyme =
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==Inhibition==
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The inhibition of pancreatic lipase has serious effects on storage and absorption of fats taken in by the body, and is therefore a potentially strong basis for pharmaceuticals to combat obesity. Because lipase is a part of the serine esterase family, it is inhibited in a similar manner. One such compound is a <scene name='Sandbox_51/Inhibitor/2'>C11 alkyl phosphonate</scene> inhibitor (shown interacting with the lipase-coplipase structure). The inhibitor acts by binding <scene name='Sandbox_51/Inhibitor_at_ser152/3'>Ser152</scene> in the active site inducing conformational changes in the beta-5 loop structures. The alkyl chain fits into the hydrophobic portion of the active lipase-colipase complex mimicking the fatty acid produced through hydrolysis in the normal enzymatic reaction <ref>Egloff, M.P., etc. "The 2.46 angstroms resolution structure of the pancreatic lipase colipase complex inhibited by a C11 alkyl phosphonate."(1995) J. Biochemistry 34: 2751-2762 [http://www.pdb.org/pdb/explore/explore.do?structureId=1lpb]</ref>. Van der waals forces between hydrophobic residues (blue) in the groove stabilize C11P binding, in addition to salt bridging and Hydrogen bonding forces with a cluster of hydrophilic residues (pick) around the Ser152 residue (purple) in the <scene name='Sandbox_51/Inhibitor_pocket/2'>inhibitor binding groove</scene>. <ref>Egloff, M., Marguet, F., Buono, G.,Verger,R.,Cambillau,C., Tilbeurgho,H. The 2.46 A Resolution Structure of the Pancreatic Lipase-Colipase Complex Inhibited by a C11 Alkyl Phosphonate? Biochemistry, 1995, 34, 275 1-2762. http://pubs.acs.org.ezproxy.messiah.edu/doi/pdf/10.1021/bi00009a003</ref>. Due to lipase's activity in the digestion and absorption of fat, there has been a growing market for lipase inhibitors for weight loss pharmaceuticals. The most popular is Orlistat (or Xenical®) which is a natural product from ''Streptomyces toxytricini'' and is the hydrogenation product of lipostation- an irreversible lipase inhibitor. This inhibitor also acts by binding Ser152, producing an ester which hydrolyzes so slow that it is practically irreversible <ref>Kordik, C., Reitz, A. "Pharmacological Treatment of Obesity: Therapeutic Strategies" Journal of Medicinal Chemistry, 1999 (42).</ref>.
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Enzymes are designed to attract and to bind specific substrates. The active site of and lysozyme and its specific ligands are described in the following sections
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==References==
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<references />
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==Mechanistic Features==
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'''Zymogen of Lysozyme: Enzymatic Precursor'''
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Zymogens are inactive enzyme precursors. Enzymes are developed in an inactive way to prevent the enzyme from digesting the cell that produced it. This process also prevents the enzyme from becoming active in the wrong portion of the body. Lysozyme's zymogen, simply titled “pre-lysozyme,” was sequenced in 1977 by R D Palmiter, J Gagnon, L H Ericsson and K A Walsh, and has since been sequenced much more extensively.
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[[Image:jrip.jpg|thumb|left|350px|Mechanism of Lysozyme]]
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<ref>Image from: http://www.google.com/imgres?imgurl=http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/pics-and-strucs/lysozyme-mech.gif&imgrefurl=http://www.vuw.ac.nz/staff/paul_teesdale-spittle/essentials/chapter-6/proteins/lysozyme.htm&usg=__ormapG4XKg-tR5GrMSOdSMTV4vE=&h=603&w=801&sz=7&hl=en&start=17&zoom=1&tbnid=nvr9gvFrUILDkM:&tbnh=143&tbnw=189&prev=/images%3Fq%3DThe%2Blysozyme%2Breaction%2Bmechanism%26um%3D1%26hl%3Den%26sa%3DN%26biw%3D1280%26bih%3D647%26tbs%3Disch:10%2C304&um=1&itbs=1&iact=hc&vpx=521&vpy=349&dur=448&hovh=191&hovw=254&tx=140&ty=48&ei=JQ_LTPKzLIjCsAPkzt2KDg&oei=IA_LTP74OsG78gapm-GFAQ&esq=2&page=2&ndsp=18&ved=1t:429,r:2,s:17&biw=1280&bih=647</ref>
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'''Mechanism'''
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The lysozyme mechanism of action results in the hydrolysis of a glycoside (hence the familial distinction of lysozyme as a glycosylase<ref>Lysozyme, 2008. Lysozyme.co.uk. http://lysozyme.co.uk/</ref>), which corresponds to the conversion of an acetal to a hemiacetal, which reaction (general degradation of glycosidic bond to units "capped" by newly formed hydroxyl groups) necessitates acid catalysis, since the conversion of acetal to hemiacetal involves the protonation of the reactant oxygen prior to actual bond cleavage. <ref>Pratt, C.W., Voet, D., Voet, J.G. Fundamentals of Biochemistry - Life at the Molecular Level - Third Edition. Voet, Voet and Pratt, 2008.</ref>. Furthermore, the transition state obtained from this protonation is a covalent, oxonium ion, intermediate that must obtain resonance stabilization. The need for some means of acid catalysis and covalent resonance stabilization is adequately provided by the Glu 35 and Asp 52 residues of lysozyme, respectively. The reaction mechanism of lysozyme is demonstrated below. In the following image, the reaction begins at the upper left-hand side, and proceeds according to reaction arrows.
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As seen to the left, lysozyme works by hydrolyzing the glycosidic bond, distorting the bond between the NAM and NAG. This produces a glycosyl enzyme intermediate, which reacts with a water molecule to produce the product and the unchanged enzyme.
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'''Active Site'''
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The <scene name='Sandbox_39/Active_site/1'>active site</scene> of lysozyme is formulated as a prominent cleft outlined by the two aforementioned catalytic amino acids, Glu 35 and Asp 52. The active site is geometrically bent to augment ligand binding, and the two amino acids interact with the ligand in the binding site. Asp52 is depicted in green, and Glu35 is depicted in purple.
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<applet load='1hew' size='350' frame='true' align='right' caption='' />
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==Binding==
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'''Ligands'''
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A <scene name='Sandbox_39/Ligands_1/1'>ligand</scene> is able to bind to the active site of an enzyme to form a biologically relevant complex. The model to the right shows a space-filling model of lysozyme with the protein distinguishable in brown and the ligand distinguishable in green. Another model of the ligand can be seen in this
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<scene name='Sandbox_39/Ribbon_ligand/1'>ribbon diagram</scene>, with the ligand protruding as a space-filling model from the active site. Here, it is clear that the ligand is a polysaccharide.
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The lysozyme reaction is characterized by hydrolysis of the beta (1-4) glycosidic bond between NAM and NAG. Lysozyme has a very specific active site, which can bind only six sugar rings from a polysaccharide chain. Once lysozyme binds to this chain, it hydrolyzes them. These six sugar rings represent the ligand of lysozyme. The lysozyme then distorts the fourth sugar in the six-membered complex, producing stress on the molecule and breaking the glycosidic bond.
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The amino acid side-chains Glu35 and Asp52 are critical to the activity of this enzyme. Glu35 acts as a proton donor to the glycosidic bond, cleaving the C-O bond in the substrate, and Asp52 acts as a nucleophile to generate a glycosyl enzyme intermediate. The glycosyl enzyme intermediate then reacts with a water molecule to give the product of hydrolysis.
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'''Inhibitors'''
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Lysozyme is best inhibited by small saccharides which act competitively with the natural substrate. The smaller saccharides will bind to the first three binding sites of the cleft (sites A-C), but will not reach sites D and E, where the enzyme cuts the glycosidic bond. So, the competitive inhibitor will stick in the cleft, not allowing the substrate to bind to the enzyme complex.<ref>http://mcdb-webarchive.mcdb.ucsb.edu/sears/biochemistry/tw-enz/lysozyme/HEWL/lysozyme-overview.htm</ref> Several known inhibitors of lysozyme are: SDS, N-acetyl-D-glucosamine, and various alcohols and oxidizing agents.<ref>http://www.worthington-biochem.com/ly/default.html</ref>
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<applet load='1hew' size='350' frame='true' align='left' caption='' />
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= Composition and Structure of Lysozyme =
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All proteins consist of carbon, hydrogen, nitrogen, oxygen, and sulfur, as do most organic molecules. Enzymes are composed in such a way as to maximize their reactivity with their desired substrate, increasing the efficiency of biological reactions. The <scene name='Sandbox_39/Elements/1'>composition of lysozyme</scene> can be seen on the left, with the carbon atoms outlined in gray, oxygen atoms in red, nitrogen atoms in blue, sulfur atoms in yellow, and the three-letter abbreviation for the <scene name='Sandbox_39/Amino_acid_residues/1'>amino acid residues</scene> in purple.
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Lysozyme, like all proteins, also contains a <scene name='Sandbox_39/C_and_n_terminal_residues/1'> 3'C and 5'N terminal </scene>, and these can be seen by following the colors of the rainbow across the molecule. Starting at the red end, the 3' C terminal end, one can work the entire way through to the 5' N terminal end, showing the folding pattern and chain of the protein.
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== Secondary Structure ==
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Lysozyme contains five <scene name='Sandbox_38/A/2'>alpha helical</scene> regions and five regions containing <scene name='Sandbox_38/B/1'>beta sheets</scene> as displayed in this <scene name='Sandbox_38/Alphab/1'>image</scene>. Linking these secondary structures, a number of beta turns and a large number of random coils make up the remainder of the polypeptide backbone. The polypeptide backbone of lysozyme involved in the 3 antiparallel beta sheets display the beta hairpin motif of supersecondary structure. This depiction of lysozyme contains an antiparallel beta-pleated sheet, which contributes greatly to the stability of the molecule by providing the correct alignment of hydrogen bonds. Lysozyme also contains a great deal of random coil, which is seen in the white regions of the molecule.
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==Amino Acid Residues==
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The amino acids present in the lysozyme polypeptide sequence have a direct influence not only on primary structure, but also on the secondary and tertiary structures, which can be influenced by polarity and charge of the sidechains. The various amino acid <scene name='Sandbox_38/Aminoi/1'>residues</scene> differ in their properties because of the great variety of side chains present on each amino acid. Polar and nonpolar (and charged and uncharged) side chains lead to various degrees of hydrophobicity and hydrophilicity, which affects protein folding. In lysozyme, these <scene name='Sandbox_38/Sc/1'>side chains</scene> are displayed for each amino acid residue.
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= Bonding Interactions =
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'''Disulfide Bonding in Lysozyme'''
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Lysozyme contains four <scene name='Sandbox_39/Disulfide_bonds/1'>disulfide bonds</scene> involving eight cysteine residues, which are highlighted in yellow on the left. Disulfide bonds are intramolecular forces that stabilize the tertiary structure of many proteins. Disulfide bonds are present in four locations in lysozyme: between Cys 6 and Cys 127, between Cys 30 and Cys 115, between Cys 64 and Cys 80 and between Cys 76 and Cys 94.
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'''Hydrogen Bonding'''
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In all proteins <scene name='Sandbox_39/Hydrogen_bonds/2'>hydrogen bonds</scene> are essential for stability. In this ribbon diagram, the hydrogen bonds can be seen between the secondary structures of lysozyme highlighted in orange. Since the double bonds of the alpha carbons in the main chain of lysozyme cause torsional strain, lysozyme is limited to very specific hydrogen bonding between the amino acid residues. This representation clearly shows how crucial hydrogen bonding is to help maintain the stability of the protein.
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<applet load='1hew' size='350' frame='true' align='left' caption='' />
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= Intermolecular Interactions =
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'''Hydrophobicity'''
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Lysozyme contains both hydrophobic and hydrophilic regions ( <scene name='Sandbox_39/Hydrophobicity/2'>Hydrophobicity</scene> ). The hydrophilic effect, or the desire for proteins to be at a specific position regarding water, is the single most important determinant of protein folding. These regions can be displayed with the hydrophobic regions in gray and the polar, hydrophillic regions in purple. This coloration highlights the location of these regions, showing that the majority of the hydrophobic regions are inside of the protein and that the majority of the hydrophillic regions are on the outside of the protein.
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'''Polarity'''
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The nature of the amino acid sidechains in the lysozyme polypeptide sequence leads to regions of varying hydrophobicities and polarities of the enzyme structure. The presence of certain regions of hydrophilicity and hydrophobicity is a driving force in determining protein structure when folding. The varying polarities of the side chains influence the locations of residues in the enzyme structure. Nonpolar residues appear blue, and polar residues appear red in the following <scene name='Sandbox_38/Non_polar_blue/1'>polarity</scene> display of lysozyme. Nonpolar residues will display hydrophobic tendencies occurring mostly on the interior of the enzyme while polar residues will increase in abundance on the surface of the protein in order to increase contact with the aqueous solvent so as to satisfy their hydrophilic nature. By observing a space-filled structural depiction of <scene name='Sandbox_38/Non_polar_blu/1'>lysozyme polarity</scene> with polar molecules colored red and nonpolar molecules colored blue the influence of polarity on nucleotide arrangement and protein folding is evident, with the blue (nonpolar) regions inside the red (polar) regions. The presence of <scene name='Sandbox_39/Water/1'>water</scene> interacting with the various hydrophilic residues is depicted to further display how polarity affects structure. Water is depicted as yellow, and the polar and nonpolar regions remain their respective color.
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'''Charge'''
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Charges of the various regions of the lysozyme structure display a hydrophilic nature and thus also affect the location of that region of polypeptides and the overall folding of the protein. Charged regions of the protein will display hydrophilic tendencies and therefore will most often be located on the surface of the lysozyme molecule where they can interact with the aqueous solvent. Non-charged portions will display hydrophobic tendencies and be located on the interior of the molecule. The effect of various <scene name='Sandbox_38/Rb/1'>charges</scene> on protein structure can be visualized with charged molecules represented by red anionic and blue cationic regions, and uncharged regions colored in grey. This depiction of lysozyme uses a spacefill representation of lysozyme to depict <scene name='Sandbox_38/Chargeddd/1'>charges</scene>.
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= Applications of Lysozyme =
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Since lysozyme has been widely recognized for its antibacterial and antifungal properties, it has a wide variety of uses both in biochemical and pharmaceutical applications. In molecular biology, lysozyme is often used in the alkaline-lysis procedure for extracting and isolating plasmid DNA. It is used extensively in the pharmaceutical field for destroying gram-positive bacteria, and can be used to support already-existing immune defenses to fight bacterial infections. This enzyme is particularly important for preventing bacterial diseases in infants. Because of its antibacterial properties, lysozyme can also be used in the food industry to help prevent spoilage of foods.
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= References =
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<references/>
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Current revision

Please do NOT make changes to this Sandbox. Sandboxes 30-60 are reserved for use by Biochemistry 410 & 412 at Messiah College taught by Dr. Hannah Tims during Fall 2012 and Spring 2013.


Lipase

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Contents

Introduction


Pancreatic lipase 1hpl (EC 3.1.1.3) is a an enzyme involved with the digestion and absorption of triacylglycerols (fats) in the intestine. It is secreted by the pancreas into the duodenum where it participates in the initial stages of breaking down fats into glyercol and three fatty acids. Lipase is a serine protease and a polar molecule. The mechanism of action requires Calcium ligands for stability and the coenzyme, colipase, for activation of the lipase mechanism. The hydrolysis products diffuse across enterocyte membrane and are absorbed into the blood [1]. Due to that role lipase has in digestion of fat, and the growing problem of obesity, there has been increased effort to develop lipase inhibitors for weight loss supplements.

Structure

1hpl is the horse pancreatic lipase enzyme that is thought to have a similar structure and function to the human pancreatic lipase. The of the human pancreatic lipase has not yet been published. Lipase is a dimer of two with 449 amino acid residues interacting with one each. The calcium ion shows that the molecule is located between acidic residues Arg, Asp and Glu. The enzyme has of various composition specific for certain interactions, an N-terminal (blue) and a C-terminal (red). In the , the N-terminal domain has the hydrolase alpha/beta folding structure, consisting of an 8 stranded alpha-beta sheet connected by helices. The C-terminal domain (where enzyme colipase binds) has a beta-sheet sandwich folding pattern [2]. The enzyme has 13 alpha helices (pink) and 22 beta sheets (yellow) per subunit, as displayed in the [3]. At the of the two monomers, interactions include 4 hydrogen bonds and 4 salt bridges that stabilize the dimer [4]. The molecule has a varying degree of interspersed within the protein(polar are shown in purple and hydrophobic in white) [5]. This is important because the enzyme actively digests at the lipid-water interface of the fatty micelles, requiring stability in both polar and non polar environments [6]. Within each monomer and dimer structure, the molecules are held together by disulfide bonds, hydrogen bonding, and electrostatic interactions (salt bridges). The enzyme has six covalent per monomer. Also, the stabilize the monomers and dimer of the enzyme at positively charged nitrogens (blue) in Arg and Lys, and negative oxygens (red) in Asp and Glu residues. are present within each monomer, as shown by the hydrogen bond forming residues (light gray), and the oxygen (red) and nitrogen (blue) atoms involved in the hydrogen bonding. Lipase is water soluble due to the polar residues on the surface, but has hydrophobic sequences on the interior. At enzyme activation and interaction with colipase, a confirmation change occurs to expose the more hydrophobic regions to the nonpolar lipid micelle.

Active Site

The of the lipase molecule is found in the N-terminal domain (residues 1-336) and contains a consisting of a Ser152-His263-Arg176 for the ester hydrolysis reaction (similar to that of a serine protease). The catalytic triad and regions around it are thought to be the best conserved aspects of lipase throughout the lipase family. The active site is covered by a 25-residue helical 'lid' blocking the binding site [7]. in complex with a triacylglyceride shows the unique lid (yellow) and beta-5 loop (pink) essential to catalysis. The 10 residue beta-5 loop changes confirmation when colipase binds, exposing lipase's oxyanion hole and hydrophobic surface. Before colipase binding however, lipase is in the structure where the beta-5 loop (residues 76-84, in pink) and lid (residues 237-261, yellow) protect the oxyanion hole from solvent interaction. The structure is accompanied by peptide shift which increase the hydrophobic surface area initiating the reaction with the lipid. one such important shift is at residues 240-253 (in yellow), in the lid structure, very close to the active site. Also, it is evident from the image that part of the beta-5 loop interacts with colipase in the open state. The lid opening is accompanied by a change in secondary structure from a mostly beta-extended confirmation to a structure where more than half the active site is formed from alpha helices [8].

Image:F1.medium.gif [9].

Function

Image:Fatty_acid_breakdown.gif[10]

Pancreatic lipase initiates the breakdown of triacylglycerols into 1,2-diacylglycerols and fatty acids. Lipase is essential to hydrolyzing the very nonpolar triacylglycerols into more polar compounds capable of diffusing across enterocyte membranes, and eventually into circulation. The digestion of triacylglycerols is a major source of energy storage in metabolism. Consequently, lipase hydrolyzes the ester bonds of the triglycerides. Lipase, as a digestive enzyme, is soluble in water but the triacylglycerides it hydrolyzes are not. In the duodenum, bile salts initiate lipase's mechanism by emulsification of the fatty acids. Bile salts are usually sodium with the deprontated form of digestive acids (mostly derivatives of cholic acid). Bile salts are stored by the gallbladder and released into the duodenum where they facilitate the formation of micelles. Bile salts or acids are amphipathic. Therefore, the hydrophobic region surrounds the fatty acids and the hydrophilic remains on the outside in the process of emulsification, creating smaller micelle molecules. Micelle formation is essential to maximize the efficiency of lipase by increasing the hydrophilic surface area on which lipase will work [11]. For the hydrolysis reaction, lipase activity requires bile acids, its coenzyme (colipase), and calcium ions to achieve the correct orientation to hydrolyze the fats [12].

Lipase

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Mechanism

In addition to the effects of bile salts, Lipase is activated by the coenzyme colipase, which binds to the C-terminal non-catalytic domain of lipase. Upon binding, active lipase is stabilized for the hydrophobic interaction with the triacylglycerides [13]. Colipase must be present for activation of lipase and acts as a bridge between lipase and the lipid [14]. Colipase is also secreted in the pancreas, but in its inactive form, which must be activated by trypsin before interacting with lipase. Colipase is a small protein cofactor with 5 conserved disulfide bonds [15], and 2 surfaces- a hydrophilic surface (site of lipase C-terminal interaction) and a hydrophobic surface (contains multiple hydrophobic loops to bridge the lipid)[16]. Colipase and lipase are opposite of the active site on the C-terminal (contacts are regions of pink and yellow, with water molecules shown in darker blue). The enzymes are bound by polar interactions such as and van der waals forces [17]. Specifically, interactions at the include bonding between residues Glu15 and Arg38 with the lipase polar lid; and Arg44, Glu45, Glu64, Arg65, and Asn89 residues with main chain, C-terminal lipase residues [18].

Lipase activation at the lipid-water interface of triacylglycerides, in the presence of colipase and bile salts, is known as interfacial activation. For the hydroloysis reaction to take place, colipase anchors lipase to the lipid-water membrane of the micelle and a surface change occurs on lipase. Colipase's 4 hydrophobic loops interact with the hydrophobic atmosphere of the triacylglyceride initiating the lipase active site binding to the lipid, and lid opening to reveal a more hydrophobic environment for the triacylglycerol. Once colipase is bound, lipase initiates a serine-like hydrolysis involving the His-Asp-Ser active site residues in the catalytic triad [19]. In the reaction, serine attacks the ester, forming an acyl-enzyme intermediate. The His and Asp residues help to stabilize the oxyanion intermediate through hydrogen bonding. Water enters the active site and reacts to release lipase and free the fatty acid. This acylation and deacylation reaction is usually completely reversible.

Lipase Triacylglyceride Hydrolysis Mechanism Image:F6.medium.gif [20].

Lipase

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Inhibition

The inhibition of pancreatic lipase has serious effects on storage and absorption of fats taken in by the body, and is therefore a potentially strong basis for pharmaceuticals to combat obesity. Because lipase is a part of the serine esterase family, it is inhibited in a similar manner. One such compound is a inhibitor (shown interacting with the lipase-coplipase structure). The inhibitor acts by binding in the active site inducing conformational changes in the beta-5 loop structures. The alkyl chain fits into the hydrophobic portion of the active lipase-colipase complex mimicking the fatty acid produced through hydrolysis in the normal enzymatic reaction [21]. Van der waals forces between hydrophobic residues (blue) in the groove stabilize C11P binding, in addition to salt bridging and Hydrogen bonding forces with a cluster of hydrophilic residues (pick) around the Ser152 residue (purple) in the . [22]. Due to lipase's activity in the digestion and absorption of fat, there has been a growing market for lipase inhibitors for weight loss pharmaceuticals. The most popular is Orlistat (or Xenical®) which is a natural product from Streptomyces toxytricini and is the hydrogenation product of lipostation- an irreversible lipase inhibitor. This inhibitor also acts by binding Ser152, producing an ester which hydrolyzes so slow that it is practically irreversible [23].

References

  1. ↑ Voet, D.,etc. "Fundamentals of Biochemistry: Life at the Molecular Level" John Wiley and Sons, Inc: New Jersey, 2008.
  2. ↑ Horse pancreatic lipase...
  3. ↑ Egloff, M.P., etc. "The 2.46 angstroms resolution structure of the pancreatic lipase colipase complex inhibited by a C11 alkyl phosphonate."(1995) J. Biochemistry 34: 2751-2762 [1]
  4. ↑ "HORSE PANCREATIC LIPASE. THE CRYSTAL STRUCTURE AT 2.3 ANGSTROMS RESOLUTION." http://bisc.cse.ucsc.edu/pages/BiscHom/1hpl_AB.html
  5. ↑ Bourne, Y., etc. "Horse pancreatic lipase..."(1994) J.Mol.Biol. 238: 709-732 [2]
  6. ↑ Fundamentals of Biochemistry...
  7. ↑ Fundamentals of Biochemistry...
  8. ↑ Thomas, A. etc. "Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity", The Journal of Biological Chemistry, 2005 September 22; 270 (48): 40074-40083.
  9. ↑ "Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity"...
  10. ↑ http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb2/part1/lipoprot.htm
  11. ↑ Bowen, R. Absorption of Lipids. 8 August 2007. 3 March 2012. <http://www.vivo.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.html>.
  12. ↑ Fundamentals of Biochemistry...
  13. ↑ Fundamentals of Biochemistry...
  14. ↑ Crandall,W., Lowe, M. "Colipase Residues Glu64 and Arg65 Are Essential for Normal Lipase-mediated Fat Digestion in the Presence of Bile Salt Micelles" Journal of Biological Chemistry, 2001, (276) 12505-12512
  15. ↑ "Colipase". Wikipedia: The Free Encyclopedia. 5 July 2011 [3]
  16. ↑ "Colipase Residues..."
  17. ↑ van Tilbeurgh H, etc."Structure of the pancreatic lipase-procolipase complex", 1992 Sep 10;359(6391):159-62. PMID:1522902.[4]
  18. ↑ "Colipase Residues..."
  19. ↑ Fundamentals of Biochemistry...
  20. ↑ Reetz, Manfield F. Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications. PNAS: 12 April 2004 [5]
  21. ↑ Egloff, M.P., etc. "The 2.46 angstroms resolution structure of the pancreatic lipase colipase complex inhibited by a C11 alkyl phosphonate."(1995) J. Biochemistry 34: 2751-2762 [6]
  22. ↑ Egloff, M., Marguet, F., Buono, G.,Verger,R.,Cambillau,C., Tilbeurgho,H. The 2.46 A Resolution Structure of the Pancreatic Lipase-Colipase Complex Inhibited by a C11 Alkyl Phosphonate? Biochemistry, 1995, 34, 275 1-2762. http://pubs.acs.org.ezproxy.messiah.edu/doi/pdf/10.1021/bi00009a003
  23. ↑ Kordik, C., Reitz, A. "Pharmacological Treatment of Obesity: Therapeutic Strategies" Journal of Medicinal Chemistry, 1999 (42).
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