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| - | {{Template: Oberholser Sandbox Reservation}}
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| - | =='''Lipase'''==
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| - | PDB ID: 1HPL<p>
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| - | </p>E.C.: 3.1.1.3<p>
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| - | </p>Number of Amino Acid Residues: 449<p>
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| - | </p>Number of Chains: 2<p>
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| - | </p>Weight: 50kDa<p></p>
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| - | ==Introduction==
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| - | The lipase class of enzymes are known to cut a lipid substrate at a specific location on their glycerol backbone. Lipase catalyzes the lipid breakdown through the hydrolysis of the esters in fatty acids.<ref>1HPL PDB SUM [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1hpl&template=main.html]</ref> While lipase is found primarily in the human pancreas, lipase can also be located in other areas in the body such as the mouth and the stomach. Pancreatic lipase, in particular, serves in human digestion to break down fats from the human diet. This lipase, therefore, is found in the digestive system of humans and is involved in the conversion of triglycerides to monoglycerides and free fatty acids. It is made by the pancreas and secreted into the duodenum, in which it will serve to break down fats.<ref>Voet, D., et al. "Fundamentals of biochemistry: life at the molecular level." John Wiley & Sons, Inc. Hoboken, 2008.</ref><p>
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| - | Human pancreatic lipase distinguishes itself from other pancreatic enzymes because when it is synthesized it is done so in its final form, without needing to be activated through proteolytic cleavage.<ref>"Pancreatic lipase." Wikipedia: the free encylopedia, 7 Nov 2011 [http://en.wikipedia.org/wiki/Human_pancreatic_lipase]</ref> However, while lipase does not need outside activation, it is not truly efficient without the presence of colipase in the duodenum.</p><p>
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| - | The crystal structure of human pancreatic lipase is still yet to be determined and the research goal of many current scientists. Therefore, the structure examined here, 1HPL, is in actuality horse pancreatic lipase, thought to have a very similar structure as well as function with regard to human pancreatic lipase.<ref>Bourne, Y., et al. ""Horse pancreatic lipase. The crystal structure defined at 2.3 A resolution"(1994) J.Mol.Biol. 238: 709-732 [http://www.pdb.org/pdb/explore/explore.do?structureId=1HPL]</ref>
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| - | </p>
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| - | ==Structural Aspects of Lipase ==
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| - | <StructureSection load='1hpl' size='500' side='right' caption='Lipase (PDB entry [[1hpl]])' scene=''>
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| - | The <scene name='Sandbox_44/Structure_of_lipase/1'>structure of lipase</scene> displays the enzyme in its natural form, with its two identical chains, each consisting of 449 amino acid residues.<ref>Bourne, Y., et al. "Horse pancreatic lipase..."</ref> The <scene name='Sandbox_44/Secondary_structure/3'>secondary structures</scene> of human pancreatic lipase consists of 30% beta sheets (shown in orange), and 22% alpha helices (shown in fuschia).<ref>Bourne, Y., et al. "Horse pancreatic lipase..."</ref> Here, beta sheets are depicted as planks, and alpha helices are shown as rockets. The remaining 48% of the enzyme's secondary structure consists of ordered, nonrepetitive sequence structure (shown in white) in contrast to the alpha helices and beta sheets. This even distribution of 48% ordered nonrepetitive structure to 52% alpha helix/beta sheet structure correlates to the appearance of the structure of lipase, which appears even to the casual observer to be about half alpha helix/beta sheet structure, and half ordered nonrepetitive structure depicted through the looping connective lines. This secondary structure of the enzyme, however, is formed due to the nature of the hydrogen bonding in between the main chains of lipase. These <scene name='Sandbox_44/Hydrogen_bonding/1'>hydrogen bonds</scene> can be seen throughout the structure of lipase, displayed here in bright yellow for clarity. In addition, pancreatic lipase has a total of twelve <scene name='Sandbox_44/Disulfide_bonds/4'>disulfide bonds</scene> in one molecule (6 disulfide bonds each per chain). As in all biological molecules, these disulfide bonds occur between Cysteine residues along the molecule. The cysteine residues involved in these disulfide bonds (shown here in red) are Cys 4 and Cys 10, Cys 90 and Cys 101, Cys 237 and Cys 261, Cys 285 and Cys 296, Cys 299 and Cys 304, and Cys 433 and Cys 449. Also serving for stabilization are numerous salt bridges throughout the lipase molecule.<p>
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| - | There are two main chains in human pancreatic lipase, shown here as Chain A and Chain B. <scene name='Sandbox_44/Chain_a/1'>Chain A</scene> (in blue) is exactly identical to <scene name='Sandbox_44/Chain_b/1'>Chain B</scene> (in green), and each chain has two domains which can be identified through <scene name='Sandbox_44/N-c_rainbow/1'>N terminus to C terminus labeling</scene>. This rainbow labeling displays the N-terminus domain of each chain in blue, leading in a color spectrum fashion to the C-terminus domain of each chain in red. These two sections of each chain are not identical in composition, however, as the N-terminus 337 residues long, comprised mainly in a 3 layer sandwich known as alpha, beta, alpha sandwich. The C-terminus, in comparison, contains a mere 112 residues that are ordered primarily in beta sandwich fashion.<ref>1HPL PDB SUM</ref> The C-terminus of lipase is where its enzyme colipase binds.<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> </p>
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| - | <p>The <scene name='Sandbox_44/Active_site_chain_a/1'>active site of Chain A</scene> is seen here highlighted in yellow, located within the N-terminus in residues 1-336. This active site contains a catalytic triad of Ser 152, His 263, and Asp 176 that facilitate the ester hydrolysis reaction carried out by lipase. This catalytic triad is very similar to that in a serine protease enzyme.<ref> Voet, D., et al. "Fundamentals of biochemistry"</ref> However, if no lipid micelles are present for digestion, the active site of lipase (containing this catalytic triad of Ser, His, and Asp) is covered with a "lid" composed of 25 residues in a helical fashion.</p><ref> Voet, D., et al. "Fundamentals of biochemistry</ref> | + | |
| - | <p>The identical <scene name='Sandbox_44/Active_site_2/1'>active site of Chain B</scene> is shown here in red, also facilitating the reaction, the mechanism of which will be discussed below.
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| - | </p>
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| - | <p> Each of the two chains of pancreatic lipase interact with one calcium ligand. A close look at the <scene name='Sandbox_44/Contacts_of_calcium/1'>contacts of calcium</scene> shows these calcium ligands to be located between the acidic residues Glu, Arg, and Asp. These four residues (Glu 187, Arg 190, Asp 195, and Asp 192) interact specifically with the calcium ligand in each chain.
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| - | </p>
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| - | Within the lipase molecule there are various hydrophilic (polar) and hydrophobic residues to account for the molecules amphiphilic properties, where stability within the molecule in both polar and non polar environments is of utmost importance. All of triacylglycerol digestion occurs at lipid-water interfaces; therefore, it is easy to understand why lipase must be stable in either environment.<ref>Voet, D., et al. "Fundamentals of biochemistry"</ref> The <scene name='Sandbox_44/Hydrophobic_residues/1'>hydrophobic residues</scene> of pancreatic lipase can be seen here in grey, while the polar residues of pancreatic lipase are shown in purple. Again, a fairly even distribution of polar to nonpolar residues are seen so as to most effectively stabilize the molecule under whatever conditions are encountered in digestion.
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| - | <p>
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| - | </StructureSection>
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| - | ==Function of Lipase==
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| - | As mentioned above, lipase serves to catalyze the hydrolysis of triacylglycerols into 2-monoacylglyercols and fatty acids.<ref>1HPL PDB SUM</ref> This breakdown of triacylglycerols in digestion is an enormous means of energy storage, and is extremely useful in human metabolism as fats from our diet are broken down. <p>
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| - | Lipase must be at the precise orientation in order to digest fats; this requires several factors to be in order for proper triacylglycerol breakdown. First, the calcium ion ligand must be present; second, the coenzyme of lipase (colipase) must be present and in the proper orientation; and finally, bile salts must be at the ready to aid in the process of breaking down fats.</p>
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| - | <p>The triacylglycerols in their initial form are not soluble in water, yet pancreatic lipase is very much water soluble, and therefore will digest these triacylglycerols at lipid-water interfaces where both are content, so to speak. How fast this digestion of triacylglycerols occurs depends largely on the size of the lipid-water interface; they are indirectly proportional to each other. Bile salts facilitate increased rates of triacylglycerol digestion in the intestine as they emulsify and solubilize fat globules. These bile salts, then, surround the globules of fatty acids and work to make them more soluble for breakdown.<ref>Voet, D., et al. "Fundamentals of biochemistry"</ref> </p>
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| - | ==Mechanism of Lipase==
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| - | <StructureSection load='1hpl' size='500' side='left' caption='Lipase (PDB entry [[1hpl]])' scene=''>
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| - | The activity of bile salts as described above are simply not enough for pancreatic lipase to function properly and efficiently; rather, lipase requires the aid of its coenzyme colipase<ref>van Tilbeurgh, H., et al. "Structure of the pancreatic lipase procolipase complex"</ref>, which binds to the <scene name='Sandbox_44/C_terminal_contact/1'>C-terminal domain of lipase</scene>(residues 337-449). This binding of colipase to lipase occurs opposite of the active site on the C-terminal, and contacts between them include Van der Waals forces and other polar interactions like salt bridges in order to achieve maximum stability between enzyme and coenzyme.<p>
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| - | When colipase binds to the nonactive site of the C-terminal domain of lipase, then, the lipase enzyme is stabilized enough so that it may proceed with any hydrophobic interactions between itself and the triacylglycerides. This binding of colipase essentially activates lipase so that it can effectively break down fats in the body; the association of colipase and lipase is what makes lipase a true catalyst.</p>
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| - | <p>When lipid micelles are present, the 25-residue "lid" over the active site undergoes a conformational changes in order that the active site may be exposed. At the same time, the beta 5 loop (consisting of 10 residues) also undergoes a conformational change to make an oxyanion hole for the enzyme, thereby creating a hydrophobic surface close to the active site entryway. This beta 5 loop had previously (before the coenzyme's binding) served to protect the oxyanion hole, but now it can reveal the hole for the reaction to proceed.<ref>Lennens, M. & Lowe, Mark. "A surface loop covering the active site of human pancreatic lipase influences interfacial activation and lipid binding." 14 Oct, 1994 The Journal of Biological Chemistry, 269, 25470-25474. [http://www.jbc.org/content/269/41/25470.full.pdf+html]</ref> As colipase binds, it forms three additional hydrogen bonds to the recently opened "lid" of the active site so as to most effectively stabilize the lid in its open position.<ref>Eydoux, C., et al. "Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation." 15 Aug, 2008. DOI: 10.1021/bi8005576 [http://pubs.acs.org/doi/abs/10.1021/bi8005576]</ref> After the binding of colipase, lipase completes a hydrolysis very similar to that of a serine protease through the use of its catalytic triad. This hydrolysis eventually releases the desired broken-down lipid products.</p>
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| - | <p>Chemically, the mechanism of triacylglycerol by lipase is achieved only through the reactions of the His, Ser, and Asp residues of the active site, as well as with aid from the stabilizing effects of nearby Phe 77 and Leu 153 found in the oxyanion hole.</p>
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| - | <p>[[Image:lipase.gif]]<ref>Reetz, M. "Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications." 20 Apr 2004, doi: 10.1073/pnas.0306866101 [http://www.pnas.org/content/101/16/5716.full]</ref></p>
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| - | <p>Mechanistically, the reaction begins as Asp abstracts a proton from the His residue, which in turn acts as a base by deprotonating Ser. This deprotonation allows Ser to attack the carbonyl of the substrate group, which then shifts its electrons in a manner so that its oxygen now has a negative charge, generating a covalent bond between the carbonyl cation and Ser. This formation is what is referred to as the "oxyanion" intermediate, a tetrahedral intermediate with a negative charge on the central oxygen. It is at this step that Phe 77 and Leu 153 stabilize the oxyanion intermediate in its oxyanion hole. However, the carbonyl soon is reformed, which breaks the bond and generates the acyl enzyme intermediate and an alcohol group. This acyl enzyme intermediate is, in turn, attacked at its carbonyl carbon, leading to the production of a regenerated Ser residue and a fatty acid, hence displaying the mechanism by which lipase cuts a large triacylglyceride into significantly smaller fatty acids.</p>
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| - | </StructureSection>
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| - | ==Applications==
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| - | <StructureSection load='1hpl' size='300' side='right' caption='Lipase (PDB entry [[1hpl]])' scene=''>
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| - | Since human pancreatic lipase plays such a vital function on the breakdown and storage of fats in the human body, there are no doubt a wealth of applications stemming from lipase manipulation. The inhibition of lipase, in particular, has been studied in great detail by pharmaceutical companies, as when lipase in inhibited, it cannot properly store and absorb fat. If the human body cannot store fat properly, it likely will lose weight- naturally a result upon which pharmaceutical companies can capitalize today in our obese society! <p>
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| - | One particular such <scene name='Sandbox_44/Inhibitor/1'>lipase inhibitor</scene> under research is a C11 alkyl phosphonate compound that inhibits lipase through its binding of Ser 152 in the active site. <ref>1HPL PDB [http://www.pdb.org/pdb/explore/explore.do?structureId=1LPB] </ref> The inhibitor fits into a hydrophobic groove within the molecule and thus is believed by scientists to be a mimic of a fatty acid which would have been produced from the binding of a triacylglyceride and the enzyme. Such devious maneuvers by lipase inhibitors may truly be the face of tomorrow's weight-loss drugs as they prevent the storage and absorption of fats by the body. Of course, caution must naturally be taken with the promise of such drugs, as a complete lack of fat absorption and/or storage by the body could be potentially fatal.</p>
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| - | <p>There are also significant clinical applications of pancreatic lipase, as we know it to be in very low concentration in serum in the duodenum. If something goes wrong in the pancreas and it begins to behave irregularly, such as is the case in pancreatitis and/or pancreatic adenocarcinoma, the concentration of pancreatic lipase in the duodenum will be extreme, as the pancreas may initiate an autolysis, resulting in far more secretion of pancreatic lipase into the duodenum than is usual, necessary, or healthy. Since this is a dangerous condition, careful monitoring of the serum concentration of lipase in the pancreas is a useful way to diagnose pancreatitis.<ref>"Pancreatic lipase." Wikipedia, the free encyclopedia</ref></p>
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| - | </StructureSection>
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| - | ==References==
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| - | <references />
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