Sandbox 44

spinqualitylabelsall models Lipase (PDB entry 1hpl) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

The displays the enzyme in its natural form, with its two identical chains, each consisting of 449 amino acid residues.^[5] The of human pancreatic lipase consists of 30% beta sheets (shown in orange), and 22% alpha helices (shown in fuschia).^[6] 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 can be seen throughout the structure of lipase, displayed here in bright yellow for clarity. In addition, pancreatic lipase has a total of twelve 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.

There are two main chains in human pancreatic lipase, shown here as Chain A and Chain B. (in blue) is exactly identical to (in green), and each chain has two domains which can be identified through . 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.^[7] The C-terminus of lipase is where its enzyme colipase binds.^[8]

The 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.^[9] 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.

^[10]

The identical is shown here in red, also facilitating the reaction, the mechanism of which will be discussed below.

Each of the two chains of pancreatic lipase interact with one calcium ligand. A close look at the 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.

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.^[11] The 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.

spinqualitylabelsall models Lipase (PDB entry 1hpl) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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^[14], which binds to the (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.

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.

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.^[15] 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.^[16] 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.

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.

Image:Lipase.gif^[17]

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.

spinqualitylabelsall models Lipase (PDB entry 1hpl) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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!

One particular such under research is a C11 alkyl phosphonate compound that inhibits lipase through its binding of Ser 152 in the active site. ^[18] 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.

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.^[19]