Sandbox 45

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Lipase is a hydrolase that catalyzes the breakdown of lipids by hydrolyzing the esters of fatty acids. Lipases are important in digestion, promoting absorption of fats in the intestines. Lipase is primarily found in the pancreas but is also found in the mouth and the stomach. Pancreatic lipase (PDB ID: 1HPL) which is pictured below is a carboxylic ester hydrolase. It is also commonly called pancreatic triacylglycerol lipase and its enzyme class number is E.C. 3.1.1.3 <ref name="1HPL PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1hpl&template=main.html] 1HPL PDB SUM </ref>. The reaction catalyzed by this enzyme is shown below. [[Image:Picture 1.png]]

 

 

<applet load='1HPL' size='400' frame='true' align='right' scene='Sandbox_45/Pancreatic_lipase/1' caption='Horse Pancreatic Lipase at 2.3 Angstroms Resolution (PDB ID: 1HPL)' />

 

The two identical chains of pancreatic lipase are shown. These two chains associate solely by <scene name='Sandbox_45/Subunit_contacts/1'>van der waals forces</scene>, a type of nonbonded contact <ref name="1HPL PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1hpl&template=main.html] 1HPL PDB SUM </ref>. Pancreatic lipase contains two <scene name='Sandbox_45/Domains/1'>domains</scene> in each chain. The N-terminal domain (red) is 337 residues long and consists mainly of 3 layer (alpha, beta, alpha) sandwich. The C-terminal domain (yellow) is 112 residues long and consists primarily of beta sandwich <ref name="1HPL PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1hpl&template=main.html] 1HPL PDB SUM </ref>. The N-terminal and C-terminal domains can be better visualized by a <scene name='Sandbox_45/Pancreatic_lipase/2'>rainbow ribbon diagram</scene>. In this representation, the N-terminus begins as blue and as it approaches the C-terminus, it changes color until it reaches red. Pancreatic lipase has a <scene name='Sandbox_45/Secondary_structure/1'>secondary structure</scene> consisting of 22% alpha helices, which are shown in blue, and 30% beta sheets, which are shown in bright green <ref name= "1HPL PDB">[http://www.pdb.org/pdb/explore/explore.do?structureId=1HPL] 1HPL PDB</ref>. The rest of the secondary structure consists of ordered, nonrepetitive structure. The secondary structure is formed due to <scene name='Sandbox_45/Secondary_structure/2'>hydrogen bonding</scene> (yellow) within the main chains. <scene name='Sandbox_45/Hydrogen_bonding/1'>Hydrogen bonding</scene> is also present in side chains to stabilize tertiary structure. In addition to hydrogen bonding, pancreatic lipase also contains twelve disulfide bonds, six in each chain, to stabilize secondary and tertiary structure. These disulfide bonds occur between two <scene name='Sandbox_45/Disulfide_bonds/6'>cysteines</scene> in the chains. Bonds occur between 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<ref name="1HPL PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1hpl&template=main.html] 1HPL PDB SUM </ref>. Many <scene name='Sandbox_45/Salt_bridges/1'>salt bridges</scene> are also seen stabilizing the structure of pancreatic lipase.

 

One <scene name='Sandbox_45/Calcium_ions/2'>calcium ion</scene> per chain, shown as a bright green ball, can be seen interacting with the protein. The calcium ions interact with <scene name='Sandbox_45/Calcium_interaction/1'>four residues</scene> in each chain. They are Glu 187, Arg 190, Asp 192, and Asp 195 <ref name="1HPL PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1hpl&template=main.html] 1HPL PDB SUM </ref>. When the rest of the protein is not visible, <scene name='Sandbox_45/Calcium_ion_interaction/4'>interaction</scene> can be seen more clearly. Because the calcium ions interact with the enzyme so far away from the active site, they do not play a role in catalysis but rather have a structural function. They function to stabilize the enzyme by aiding it in maintaining its three-dimensional shape, especially when it is in the active conformation and is exposed to high temperatures <ref name= "Identification of a Calcium Binding Site in Staphylococcus hyicus Lipase:

 

In pancreatic lipase, there is a relatively equal distribution of <scene name='Sandbox_45/Hydrophobic_residues/5'>hydrophobic residues and hydrophillic residues</scene>. Hydrophobic residues are shown in red and hydrophillic residues in blue when clicking the green link. The space fill model is another useful representation of the distribution of <scene name='Sandbox_45/Space_fill_hydrophobic/1'>hydrophobic and hydrophillic residues</scene>. When the hydrophillic residues are removed and only <scene name='Sandbox_45/Hydrophobic_core/1'>hydrophobic residues</scene> are shown, it is clear that the core of the enzyme is made of hydrophobic residues while the hydrophillic residues are mainly located on the surface of the enzyme. <scene name='Sandbox_45/Acidic_and_basic_residues/2'>Acidic and basic residues</scene> contribute to the relative hydophilicity of the protein since they can be protonated and deprotonated at varying pH levels, causing a charge to be present. The distribution and number of acidic (red) and basic (blue) residues is relatively even. White coloring represents nonpolar residues.

 

Pancreatic lipase contains an identical active site in each of the two identical chains. There are three important active site residues that form the <scene name='Sandbox_45/Catalytic_triad/3'>catalytic triad</scene>. They are highlighted in yellow. These three catalytic residues are all located in the N-terminal domain and consist of Ser 152, Asp 176, and His 263 <ref name="1HPL PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1hpl&template=main.html] 1HPL PDB SUM </ref>. Two other residues, <scene name='Sandbox_45/Catalytic_triad/4'>Phe 77 and Leu 153</scene> (highlighted in blue), are also important and act in the oxyanion hole to stabilize the negatively charged tetrahedral intermediates formed during catalysis <ref name= "EMBL-EBI">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/CSA/CSA_Site_Wrapper.pl?pdb=1HPL] EMBL-EBI </ref>. Histidine begins the reaction by acting as a base (the negative charge interaction of aspartate makes histidine a better base) in order to deprotonate serine so that it can attack the carbonyl carbon of the ester. This results in the formation of a covalent bond between serine and the carbonyl carbon. This forms a negatively charged tetrahedral intermediate which is stabilized by Phe 77 and Leu 153 in the oxyanion hole. The carbonyl then reforms, breaking the ester bond, and histidine protonates the oxygen containing leaving group. The carbonyl is once again attacked but this time by water which is deprotonated by histidine. Another negatively charged tetrahedral intermediate is formed which is stabilized in the oxyanion hole. The carbonyl carbon is again reformed but this time serine is released as the leaving group and protonated by histidine as it leaves, regenerating the enzyme so it can catalyze another reaction and releasing the cleaved lipid product <ref name= "EMBL-EBI">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/CSA/CSA_Site_Wrapper.pl?pdb=1HPL] EMBL-EBI </ref>. In the mechanism pictured below, Asp is the acting catalytic residue, not Glu, although Glu residues also commonly participate in ester hydrolysis reactions as well. The general amino acid pictured as stabilizing the negatively charged tetrahedral intermediate in the oxyanion hole can actually be thought of as Phe 77/Leu 153.

 

Pancreatic lipase is shown here in a complex with the 10 kDa coenzyme colipase <ref name= "A cross-linked complex between horse pancreatic lipase and colipase">[http://www.sciencedirect.com/science/article/pii/0014579389815923] A cross-linked complex between horse pancreatic lipase and colipase</ref><ref name= "1LPB PDB">[http://download.rcsb.org/pdb/explore/explore.do?structureId=1LPB] 1LPB PDB</ref>. Colipase must be in association with lipase for it to have catalytic function. Without colipase present, the accumulation of amphiphiles at the oil/water interface in the duodenum would prevent pancreatic lipase from binding. Binding of colipase also causes the "lid" which covers the active site to be lifted off of it, exposing the active site for catalysis, in addition to some other small conformation shifts within lipase <ref name= "Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity">[http://www.jbc.org/content/280/48/40074] Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity</ref>. The structure of colipase is similar to lipase in the fact that it has two identical chains, although it is much smaller. Association between colipase and lipase occurs at the C-terminal end of the lipase chains <ref name= "The lipase/colipase complex is activated by a micelle: neutron crystallographic evidence">[http://www.sciencedirect.com/science/article/pii/S000930849800036X] The lipase/colipase complex is activated by a micelle: neutron crystallographic evidence</ref>. The colipase and lipase chains associate by 11 hydrogen bonds, involving <scene name='Sandbox_45/Hydrogen_bonding_colipase/1'>14 different residues</scene>. There are also <scene name='Sandbox_45/Van_der_waals_between_c_and_l/1'>98 van der waals interactions</scene>, involving 33 residues, which function to hold the colipase and lipase chains in association with one another <ref name= "1LPB PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1lpb&template=main.html] 1LPB PDB SUM</ref>.

In this structure, only one of the two identical chains is shown for lipase and colipase to better visualize the interaction of substrates and ligands with the protein. <scene name='Sandbox_45/Lipase_colipase_inhibitor/1'>Methoxyundecylphosphinic acid (MUP)</scene>, a C11 alkyl phosphonate, is a competitive inhibitor of pancreatic lipase which binds to the active site. It is highlighted in purple. There are also five B-octylglucoside molecules in association with lipase. They are shown in grey and red. MUP forms hydrogen bonds with <scene name='Sandbox_45/Inhibitor_interaction/3'>four residues</scene>: Ser 152 and His 263, which are part of the catalytic triad, and Phe 77 and Leu 153 which are the stabilizing residues located in the oxyanion hole <ref name= "1LPB PDB SUM">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1lpb&template=main.html] 1LPB PDB SUM</ref>. MUP also interacts with other nearby residues through hydrophobic <scene name='Sandbox_45/Van_der_waals/2'>van der waals</scene> interactions. MUP is shown in dark green and surrounding residues which it interacts with are shown in light green.

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