Introduction
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.
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.[1] The complete DNA sequence of HPL was determined in 1992 by combing polypeptide chain and cDNA sequencing. The most notable difference between HPL and human lipase is that Lys 373 is not conserved in HPL. The significance of this difference will be discussed in the mechanism section.[2]
Structure
Horse pancreatic lipase contains two identical peptide chains containing 449 amino acid residues. The N to C terminal order is shown where the N terminus is in blue and can be followed to the C terminus in red. Each chain contains
. 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.[3] The active site of HPL is highlighted in red to show its location in the N terminal domain of the A chain. Additionally, the can be seen here where negatively charged amino acid residues are seen in red, and positively charged amino acids are seen in blue.
The 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 shown in grey are mostly facing towards the interior of the protein. Conversely, the in pink point congregate more on the exterior and point outwards. [4]
Disulfide Bonds
Additional tertiary stability is provided by between cysteine residues shown in yellow linkages. Cysteine residues not involved in disulfide bonds are shown as spheres.
Chain Contacts
The two chains of HPL are connected through , , , and . In the last scene, water molecules are shown as pink spheres.
Calcium Ions
Two calcium ions are also present in the protein to further 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.[5] This is most likely due to reduced structural coordination. A more detailed view of the calcium coordination can be seen here:
Colipase Binding
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.[7]
When bound to colipase,HPL exists in its active, .[8] 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.
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 , as horse, ox, pig, rat, dog, and chicken colipases all can activate HPL and have similar disassociation constants. [9]
In the presence of colipase, the enzyme is activated which moves the (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.[10] The reorganization of the flap also induces a second conformational change that creates the oxyanion hole.[11]
Active Site and Mechanism
The 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.
[13]
This active site in HPL is used to hydrolyze triacylglycerol into carboxylate and diacylglycerol.
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.[15]
In a comparison of HPL and human lipase, the most notable difference is the lack of Lys 373 in HPL. In human lipase, Lys 373 hyrrolyzes p-nitrophenyl acetate in a biphasic process. This release is indicative of a fast acylation step, and proves that acylation and p-nitrophenyl acetate hydrolysis occur in separate steps. This further evidence offers support for covalent catalysis in the active site and that there is only one active site (which was not completely known in 1992 when the protein was sequenced).[16]
Ligand Binding / Inhibition
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.[17] The 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 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.[18] C11P was shown to be 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.
In addition to the C11P inhibitor bound in the active site, the crystallized structure showed (blue) that were stabilized by interactions with C11P and needed for the crystallization. [20] In total, the protein compounds contain 1 covalent inhibitor that can be present in both enantiomers, 1 Ca+ ion, , and 5 detergent molecules.
Additional known inhibitors of lipase include:
Tetrahydrolipstatin
Orlistat (Alli)
Monoacylglycerol
Wheat flower (WFL1)
Lipase Assays
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.
References
- ↑ http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb16926.x/pdf
- ↑ http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb16926.x/full
- ↑ http://www.pdb.org/pdb/explore/explore.do?structureId=1HPL
- ↑ http://www.pdb.org/pdb/explore/remediatedSequence.do?structureId=1HPL
- ↑ http://www.springerlink.com/content/g5h1613440115701/fulltext.pdf
- ↑ http://www.pdb.org/pdb/explore/viewerLaunch.do?viewerType=LX&structureId=1HPL&hetId=CA
- ↑ http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb16926.x/pdf
- ↑ http://www.pdb.org/pdb/explore/explore.do?structureId=1ETH
- ↑ http://www.sciencedirect.com/science/article/pii/S0300908481801964
- ↑ http://www.pdb.org/pdb/explore/explore.do?structureId=1ETH
- ↑ http://www.nature.com/nature/journal/v362/n6423/abs/362814a0.html
- ↑ http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0218
- ↑ http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0218
- ↑ http://www.pnas.org/content/101/16/5716/F6.expansion.html
- ↑ http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/MACiE/entry/getPage.pl?id=M0218
- ↑ http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1992.tb16926.x/full
- ↑ http://www.rcsb.org/pdb/explore/explore.do?structureId=1LPB
- ↑ http://pubs.acs.org/doi/abs/10.1021/bi00009a003
- ↑ http://www.rcsb.org/pdb/explore/explore.do?structureId=1LPB
- ↑ http://pubs.acs.org/doi/abs/10.1021/bi00009a003
