Lipase, as its name suggests, is an enzyme responsible for the cleavage of triglycerides and other types of lipid molecules. The mechanism by which it completes this task designates it as a hydrolase. There are different types of lipases, many of which work in similar ways. For instance, Human Pancreatic Lipase, or HPL, splits triglycerides, the main lipids in the human diet, into glycerol and three fatty acids. The structure shown at right is that of Horse Pancreatic Lipase [1]. It consists of two identical subunits, totaling 449 amino acids each, and totals 50 kDA. To better visualize the directionality of the subunits with respect to each other we can use a . This diagram shows the N-terminus of each subunit in blue, the follows the spectrum through green, yellow, orange, and finally the C-terminus is shown in red.
Basic Structure
The of lipase (just one subunit) include 102 residues which create 13 alpha helices, shown in red, and 139 residues involved in beta sheets totaling 28 strands, shown in gold. The alpha helices account fot 22% of the proteing, while the beta sheets comprise 30%.[2] The
can show much about the structure of the protein. The negatively charged residues in this scene are shown in a red shade, and the positively charged residues are in a dark blue. Notice how the negatively charged residues are oriented around the positive calcium ligands. These residues are represented in ball and stick model form to show their interaction with the ligands. All other residues are colored black in this scene. If we analyze together the we notice that the majority of the nonpolar residues, in green, are buried in the center of each subunit. This is a good illustration of the hydrophobic collapse that contributes to the protein folding into its tertiary structure. The polar residues, colored blue, are exposed to the aqueous environment. The tertiary stucture of the molecule is stabilized by 6 [3] and ionic interactions with a calcium ligand within each subunit. Finally, the quaternary structure is completed by the adjoining of the two identical subunits.The include hydrogen bonds, hydrophobic interactions, salt bridges, and other interactions. One particular interaction that can be seen is a salt bridge between LYS80 on subunit B and GLU370 on subunit A. As the figure shows,the interactions between subunits occur between the peripheral portions of each subunit. This leaves an empty cavity-like area in the center of the protein, mot likely the entry point for the lipid substrate.
The Calcium Ligand
The are two calcium ions, one buried within each subunit. This scene shows the interactions between the calcium ion (shown in green) in subunit A and the following residues from subunit A: GLU187, ARG190, ASP192, and ASP195. What can be seen from the figure is the ionic interactions between the calcium ion (2+ charge) and the various negatively charged amino acid residues such as aspartate and glutamate. Interestingly, even the carbonyl oxygen of the backbone on ARG190 interacts with and is stabilized by the calcium ion. In addition to interactions with these molecules, the calcium ion is also stabilized by the oxygen atoms from two water molecules shown in pink. These interactions between the amino acid residues and the ligand are crucial for proper protein folding, and subsequently protein function.
The Active Site
Image:Hydrolysis of trigly.png This is the reaction catalyzed by most lipases. Shown is a triglyceride being hydrolyzed by water, resulting in glycerol and three liberated fatty acids. Pancreatic Lipase, however, only hydrolyzes two ester bonds from triglycerides.
Lipase works with a substrate that consists usually of triglycerides. Triglycerides are composed of glycerol connected via ester bonds to three fatty acids. These molecules are split by hydrolysis (see figure)
[4]. This is the reaction catalyzed by most lipases. Lipase acts on the exterior fatty acids of triglycerides, hydrolyzing the bonds and freeing the two outer fatty acids from the glycerol backbone. The of lipase consists mainly of three residues. These three residues are SER152, ASP176, and HIS263, shown in red stick representation. The mechanism for these hydrolysis reactions begins with SER152 attacking a carbonyl carbon on the fatty acid of a triglyceride, forming a tetrahedral intermediate. When the oxyanion reforms its double bonded form, the oxygen originally involved in the ester bond to the glycerol backbone acts as the leaving group and accepts a hydrogen from HIS263. Next, water is converted into a nucleophile by donating a hydrogen to the HIS263, then attacking the new carbonyl which has been formed with serine. The SER152 now acts as the leaving group, producing a fatty acid chain which has been separated from glycerol and regenerating the enzyme active site. This mechanism is repeated for the two outer fatty acids of each triglyceride. The final product of complete hydrolysis by Lipase is two fatty acids and one monoglyceride. Seen here is the Candida rugosa lipase in with two molecules of cholesteryl linoleate (grey). The active site residues are highlighted in red. Finally, the between the cholesteryl linoleate and lipase can be seen here. Though this lipase is from a species of candida, a yeast, notice the similarities in structure to that of the horse. For specificity, this scene shows the by amino acid residue.
Evolutionary Conservation
This scale is used to identify the evolutionary conservation of certain residues. The red residues are the most conserved and the blue tend to be variable between variations of the protein.
The consistency of residues between variations of lipases can be described as the of the protein. As it can be observed from the conservation of individual residues, the residues near the active site and close to the calcium ligand have the highest average conservation. The conservation of residues becomes more variable the farther away from each active site.
Optimization of Action
In order for the action of Pancreatic Lipase to be optimized in vivo, a coenzyme called colipase must be secreted by the pancreas and activated by intestinal trypsin. Colipase prevents bile salts from inhibiting the regular action of Pancreatic Lipase. Colipase binds to the C-terminal of a subunit of Pancreatic Lipase, stabilising the active conformation. Shown here is bound to a subunit of Pancreatic Lipase. This view shows the many between colipase and Pancreatic Lipase. The pancreatic lipase - colipase complex structure and mechanism was discovered through the use of an , a C11 alkyl phosphonate. The C11 alkyl group of the inhibitor acts as a fatty acid of a triglyceride would, but cannot be cleaved from the phosphate head of the phosphonate. Seen in this scene is the lipase - colipase complex with mutliple phosphonate inhibitors.
References
- ↑ http://www.pdb.org/pdb/explore/explore.do?structureId=1hpl
- ↑ http://www.pdb.org/pdb/explore/remediatedSequence.do?structureId=1HPL#DSSPRefAnchor
- ↑ http://molvis.sdsc.edu/fgij/fg.htm?mol=1hpl
- ↑ http://www.engin.umich.edu/dept/che/research/savage/energy.html
