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| = Horse Pancreatic Lipase =
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| <StructureSection load='1hpl' size='500' side='right' caption='Structure of Horse Pancreatic Lipase (PDB entry [[1hpl]])' scene=''> | | <Structure load='1AKE' size='500' frame='true' align='right' caption='Adenylate_Kinase ' scene='Insert optional scene name here'/> |
| == Introduction ==
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| Lipase, which is produced primarily in the pancreas, functions as a catalyst in the hydrolysis of ester bonds in lipid substrates. This makes lipases an essential molecule for fat digestion.
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| Horse pancreatic lipase’s (EC 3.1.1.3) function is to convert triacylglycerols into 2-monoacylglycerols and free fatty acids. These monomers are then able to be shuttled into the small intestine to be absorbed into the lymphatic system.<ref>http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb16926.x/pdf</ref>
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| ==Structure == | | ==Description== |
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| | Adenylate Kinase, also known as ADK, is an phosphotransfer enzyme that catalyzes the reversible transfer of phosphate between ATP and AMP. It plays an important role in cell maintenance and cell growth being involved with energy metabolism, signaling, and nucleotide synthesis. The reaction that takes place during the catalysis is ATP + AMP = 2ADP. The enzyme has two conformations, where the inactive form is open, and the active form is closed. The open conformation allows substrates to bind, and the closed form is when the substrate is already bound, and the catalysis is taking place. The enzyme is found in various organisms, and the following images shows the structure of Adenylate Kinase from Yersinia pestis, also known as yeast. |
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| Horse pancreatic lipase contains two identical peptide chains containing 449 amino acid residues. The N to C terminal order is shown <scene name='Sandbox_50/5-3_rainbow/1'>here</scene> where the N terminus is in blue and can be followed to the C terminus in red. Each chain contains
| | ==Structure== |
| <scene name='Sandbox_50/N_and_c_terminus/1'>two well defined domains</scene>. The N-terminal domain is shown in blue and the C terminal domain is shown in green. The N terminal domain is characterized by an expected alpha/beta hydrolase fold, while the C terminal domain contains a beta sheet sandwich that is involved in colipase binding.<ref>http://www.pdb.org/pdb/explore/explore.do?structureId=1HPL</ref> The active site of HPL is highlighted in red to show its location in the N terminal domain of the A chain. Additionally, the <scene name='Sandbox_50/Aa_types/1'>charge distribution</scene> can be seen here where negatively charged amino acid residues are seen in red, and positively charged amino acids are seen in blue.
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| The <scene name='Sandbox_50/Helix/2'>secondary structure</scene> of HPL contains 13 alpha helices and 28 strands of beta sheets, representing 22% and 30%, respectively, of the protein's residues. Hydrophic collapse contributes to much of the secondary and tertiary structures, as the <scene name='Sandbox_50/Hphobic_residues/2'>hydrophobic residues</scene> shown in grey are mostly facing towards the interior of the protein. Conversely, the <scene name='Sandbox_50/Polar_residues/2'>polar residues</scene> in pink point congregate more on the exterior and point outwards. <ref>http://www.pdb.org/pdb/explore/remediatedSequence.do?structureId=1HPL</ref>
| | Adenylate Kinase is made up of 214 amino acids, and the <scene name='Sandbox_50/Ak_backbone/1'>backbone</scene> of the protein can be seen on the right in light blue surrounding the non-hydrolysable substrate analogue (red). |
| | The <scene name='Sandbox_50/Ak_secondary_structure/1'>secondary_structure</scene> of the protein contains 12 alpha helices (yellow) and 7 beta sheets (green). This secondary structure is held together by <scene name='Sandbox_50/Ak_hydrogen_bonds/1'>hydrogen_bonds</scene>, which are anti-parallel between the beta sheets. This hydrogen bond network also assists in the flexibility of the enzyme. |
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| === Disulfide Bonds === | | ==Hydrophobic and Hydrophilic Residues== |
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| Additional tertiary stability is provided by <scene name='Sandbox_50/Disulfide_bonds/1'>disulfide bonds</scene> between cysteine residues shown in yellow linkages. Cysteine residues not involved in disulfide bonds are shown as spheres.
| | The <scene name='Sandbox_50/Ak_hydrophobic_residues/1'>hydrophobic_residues</scene> of ADK, seen in gray, is buried in the interior of the protein. While the <scene name='Sandbox_50/Ak_hydrophiblic_residues2/1'>hydrophilic_residues</scene>, all the charged and polar side chains (purple), are on the surface of the protein and exposed. The location of the residues depend on the solvent and the environment that the protein is found in. All the hydrophobic residues aggregate together, and bury themselves in the interior of the protein to minimize their contact with their environment. The hydrophilic residues, on the other hand, is exposed on the surface because the enzyme is in an hydrophilic environment. Although, most of the hydrophilic residues would be exposed, it is possible for some of the to be buried in the interior, but they would interact with each other be stabilized there. There are also hydrophilic residues in the active site of the enzyme. |
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| === Chain Contacts === | | ==Active Site== |
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| The two chains of HPL are connected through <scene name='Sandbox_50/Hydrophobic_chain_interactions/2'>hydrophobic interactions</scene>, <scene name='Sandbox_50/Hydrogen_bonds_non_water/1'>hydrogen bonds</scene>, <scene name='Sandbox_50/Salt_bridges/1'>salt bridges</scene>, and <scene name='Sandbox_50/Putative_water_bridges/1'>putative water bridges</scene>. In the last scene, water molecules are shown as pink spheres. | | The active site, like mentioned above, is where the substrates binds to the enzyme to be catalyzed. In ADK, the <scene name='Sandbox_50/Ak_ligand_contact1/1'>ligand_contacts</scene> (gray, blue, pink), is in the interior of the protein. The pink is where the ligand binds directly. There are mostly hydrophilic residues present in the active site because water enters the active site regularly it causes the hydrophobic residues to still be buried within the protein. But there are some hydrophobic interactions that take place between the enzyme and the substrates, which helps stabilizes the substrate in the site, so that it can be catalyzed. There are six <scene name='Sandbox_50/Ak_catalytic_residues1/1'>catalytic_residues</scene>, which are highlighted black on the image, and they are specifically involved in the catalyzes of the substrates forming hydrogen bonds with the substrate. The catalytic residues are all charged residues and include Lysine, Aspartic acid, and Arginine. These residues also allow for electrostatic interactions but can be effected by the presence of the water in the active site. |
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| === Calcium Ions === | | ==Solvent== |
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| Two calcium ions are also present in the protein to further <scene name='Sandbox_50/Calcium_coordination_no_bb/2'>coordinate</scene> the structure of HPL. Each chain contains one calcium ion, each bound by the same residues. While the calcium ligands are no involved in the manipulating the substrate in the active site, enzymatic activity has been shown to be related to free calcium concentration. At lower calcium concentrations, lipases show reduced activity.<ref>http://www.springerlink.com/content/g5h1613440115701/fulltext.pdf</ref> This is most likely due to reduced structural coordination. A more detailed view of the calcium coordination can be seen here:
| | The <scene name='Sandbox_50/Ak_water6/1'>solvent</scene>, which is water (blue), can be co-crystallized with the enzyme. The water can be found all around the protein but there is also some water molecules in the active site, around the ligand. This further indicates why the hydrophilic residues are found on the surface, and the nonpolar residues are buried away. The water creates a hydrophilic environment, and the hydrophobic residues aggregate together in the interior, which is the hydrophobic effect and drives the water out. So for the most part, there are not water molecules in between the secondary structure, but there are some water molecules in the open spaces between the backbone.The hydrophilic residues in the active site allow water to be present, and also make it easier for the substrates to enter and facilitates in the catalysis. |
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| [[Image:Calcium binding.PNG|600px|center|thumb| Calcium Ion Coordination<ref>http://www.pdb.org/pdb/explore/viewerLaunch.do?viewerType=LX&structureId=1HPL&hetId=CA</ref> ]]
| | ==References== |
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| == Colipase Binding ==
| | http://www.ncbi.nlm.nih.gov/pubmed/21365689 |
| When bile salts accumulate at the lid/water interface, adsoprtion of the enzyme on its substrates is prohibited. In order to overcome this inhibitory effect, colipase binds to HPL and anchors lipase at the interface coated with bile salts.<ref>http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb16926.x/pdf</ref>
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| When bound to colipase,HPL exists in its active, <scene name='Sandbox_50/Colipase_binding/1'>open configuration</scene>.<ref>http://www.pdb.org/pdb/explore/explore.do?structureId=1ETH</ref> Here chains A and C (blue and pink) are the chians from the original HPL protein, and chains B and D (green and yellow) are colipase peptides.
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| Rate studies show that the disassociation constant of the lipase-colipase complex is 101.1x10^−9 M. When the substrate is absent, the disassociation constant increases by several orders, indicating that disassociation of colipase from lipase increases when no substrates are present. These studies confirm that pancreatic lipase has many highly <scene name='Sandbox_50/Conserved_residues/1'>conserved residues</scene>, as horse, ox, pig, rat, dog, and chicken colipases all can activate HPL and have similar disassociation constants. <ref>http://www.sciencedirect.com/science/article/pii/S0300908481801964</ref>
| | http://medical-dictionary.thefreedictionary.com/adenylate+kinase |
| {{Template:ColorKey_ConSurf_NoYellow_NoGray}}
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| In the presence of colipase, the enzyme is activated which moves the <scene name='Sandbox_50/N-terminal_flap/1'>N-terminal flap</scene> (shown in red) which is composed of amino acids 216-239. The N-terminal flap moves in a concerted fashion along with the C-terminal domain to reveal the active site (green), allowing it to bind with a substrate. It is hypothesized that this flexibility may have significance in binding the colipase-lipase complex with the water-lipid interface.<ref>http://www.pdb.org/pdb/explore/explore.do?structureId=1ETH</ref> The reorganization of the flap also induces a second conformational change that creates the oxyanion hole.<ref>http://www.nature.com/nature/journal/v362/n6423/abs/362814a0.html</ref>
| | http://en.wikipedia.org/wiki/Adenylate_kinase |
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| == Active Site and Mechanism == | | http://www.ebi.ac.uk/interpro/IEntry?ac=IPR007862 |
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| [[Image:Mech..PNG|200px|right|thumb| HPL hydrolysis reaction<ref>http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0218</ref>]]The <scene name='Sandbox_50/Active_site_and_some/2'>active site</scene> of HPL is characterized by side chain residues Ser 152, His 263, and Asp 176 shown in red. Additionally, the main chain amides of Phe 77 (blue) and Leu 153 (green) are shown, as both are also involved with enzymatic activity.<ref>http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0218</ref>
| | http://www.whatislife.com/reader/interaction-reader.html |
| This active site in HPL is used to hydrolyze triacylglycerol into carboxylate and diacylglycerol.
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| | | Voet, D., Voet, J., and Pratt, C. W. ''Fundamentals of Biochemistry: Life at the Molecular Level''. 3rd Edition. (2008) |
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| [[Image:Lipase mech.gif|200px|left|thumb| lipase-catalyzed hydrolysis of esters<ref>http://www.pnas.org/content/101/16/5716/F6.expansion.html</ref> ]]In the first step, His263 deprotonates Ser152. Ser152 is then free to attack the carboxy carbon of triacylglycerol through a nucleophilic addition reaction. Next, the diacylglycerol product is eliminated when the oxyaninion collapses. This deprotonates His263. In the third step, His263 deprotonates water, which can then attack the carboxyl carbon of Ser152 through a nucleophilic addition reaction. Finally, the carboxylate product and Ser152 are eliminated with the collapse of the oxyanion, and His263 is deprotonated.<ref>http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0218</ref>
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| == Ligand Binding / Inhibition ==
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| Because lipase is a member of the serine esterase family, it can be inhibited by serine reagents. In an effort to further characterize the mechanism and active site binding of a substrate, a C11 alkyl phosphonate (C11P) compound was synthesized and found to be an inhibitor of lipase. In this study, human pancreatic lipase was purified and porcin-activated colipase was used to activate it. The horse pancreatic lipase and human pancreatic lipase enzymes are highly conserved, so active site resides are similar.<ref>http://www.rcsb.org/pdb/explore/explore.do?structureId=1LPB</ref> The <scene name='Sandbox_50/C11_alkyl_phosphonate_in_activ/1'>crystal structure</scene> of this molecule inhibiting the activated lipase-colipase complex was determined at 2.46 A (Lipase B chain in green, colipase in blue, C11 alkyl phosphonate in red). C11P was found covalently bound at the <scene name='Sandbox_50/C11p_bound/1'>active site</scene> Ser 152 (red), and phe 77 (purple) and Leu 153 (green) amides formed hydrogen bonds with the oxygen of the phosphorus ester of C11P. This confirms mechanism speculations of the role of these residues in oxyanion stabilization. The geometry of this oxyanion hole was conserved when two other inhibitors were characterized in the active site.<ref>http://pubs.acs.org/doi/abs/10.1021/bi00009a003</ref> C11P was shown to be <scene name='Sandbox_50/C11p_bound_h_phobics/1'>further stabilized</scene> by van der Waals contacts with hydrophobic side chains Ala 178, Phe 215, Pro l80, Tyr ll4, Leu 213 (shown in blue). A similar representation of these interaction with a methoxyundecylphosphinic acid inhibitor is shown in the image below.
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| [[Image:MUP_1LPB.png|350px|center|thumb| methoxyundecylphosphinic acid interactions<ref>http://www.rcsb.org/pdb/explore/explore.do?structureId=1LPB</ref> ]]
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| In addition to the C11P inhibitor bound in the active site, the crystallized structure showed <scene name='Sandbox_50/Detergents/1'>5 octyl beta-glucoside detergent molecules</scene> (blue) that were stabilized by interactions with C11P and needed for the crystallization. <ref>http://pubs.acs.org/doi/abs/10.1021/bi00009a003</ref> In total, the protein compounds contain 1 covalent inhibitor that can be present in both enantiomers, 1 Ca+ ion, <scene name='Sandbox_50/Wate_on_1lpb/1'>293 water molecules</scene>, and 5 detergent molecules.
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| Additional known inhibitors of lipase include:
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| Tetrahydrolipstatin
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| Orlistat (Alli)
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| Monoacylglycerol
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| Wheat flower (WFL1)
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| == Lipase Assays ==
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| Current research in Dr. Tims' Chem 412 lab is focused on lipase activity in multi-enzyme dietary supplements. The presence of lipase and its activity has been tested for using a western blot analysis, colorimetric assay, and titrimetric assay. Lipase was present in all three tests on pancreatin samples. Only the titrimetric assay showed lipase activity in the multi-enzyme tablet. The titrimetric and colorimetric assays used an olive oil emulsion as a lipase substrate. Products of the previously described mechanism were detected by a decrease in fluorescent activity of the breakdown products in the colorimetric assay. The titrimetric assay took advantage of the fatty acid products of lipase and how much sodium hydroxide it took to get each reaction to a specific pH.
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| == References ==
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| <references />
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| </StructureSection>
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