Lipase catalyzes the breakdown of lipids by hydrolyzing the esters of fatty acids. Its function is important for digestion and promoting absorption of fats in the intestines. Lipase is primarily found in and secreted by the pancreas, but is also found in the saliva and stomach. Pancreatic lipase (PDB ID: 1HPL) which is pictured to the right, 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 [1]. The bile salt-stimulated lipase (BSSL) is found in breast milk. The hormone-sensitive lipase (LIPE) hydrolyzes a variety of esters. Monoacylglycerol lipase (MAGL) hydrolyzes intracellular triglycerides to fatty acid and glycerol. MAGL functions together with LIPE. The reaction catalyzed by the enzyme is shown below.
Further breakdown ultimately results in 2-monoacylglycerols and free fatty acids [2]. An in depth discussion of the mechanism can be found in the Lipase Catalytic Mechanism section. The determination of the structure and function of lipase was a gradual process. Lipase activity was first demonstrated in the pancreas by Claude Bernard in 1846. However, it wasn't until 1955 that Mattson and Beck demonstrated a high-specificity of pancreatic lipase for triglyceride primary esters [3]. In recent years, determination of the crystal structure of pancreatic lipase has become the primary focus as many scientists have worked to further this. See also
Pancreatic lipase is a 50 kDa protein. While the crystallographic asymmetric unit contains two identical chains, information (REMARK 350) in the data file 1hpl indicates that the dimer is a crystallization artifact, and that the functional form (also called the biological assembly) is a single chain (monomer). The chain consists of 449 residues [4]. The secondary structures of lipase (in 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 for 22% of the protein, while the beta sheets comprise 30%. Each chain contains two well defined domains. The N terminal domain, shown in blue, is characterized by an alpha/beta hydrolase fold. While the C terminal domain, shown in green, contains a beta sheet sandwich which interacts with colipase [5]. Each monomer and dimer structure of lipase are held together by disulfide bonds, hydrogen bonds, and electrostatic interactions (salt bridges). Lipase has 12 total disulfide bonds between cysteine residues. Salt bridges are formed between the positively charge nitrogens (blue) in Arg and Lys, and negative oxygens (red) in Asp and Glu residues. Hydrogen bonds (in yellow) also stabilize the enzyme between main chain and side chain atoms. Lipase has a distinct distribution of hydrophobic and hydrophilic residues (purple spacefill represents polar residues). Hydrophobic collapse contributes to much of the secondary and tertiary structures, as the hydrophobic core residues (shown in white) make up the interior of the protein, while polar residues (transparent blue) are on the surface [6]. In addition, lipase has two calcium ligands. One is buried in each monomer subunit. The calcium ion is essential to protein folding and enzyme activity [7]. The image shows the green calcium ion in subunit A, coordinated by Glu187, Arg190, Asp192, and Asp195 residues. The Ca(+2) charge is stabilized by negatively charged glutamate and aspartate residues, and the oxygen atoms from two water molecules (pink).
In addition, lipase has a unique lid (green) that blocks solvent from entering the active site (red). The lid is a 25-residue helical structure that protects the oxyanion hole. The lid (yellow) is especially important to substrate binding as it undergoes a dramatic shift that alters the secondary structure of the lipase binding site from a closed lid structure (active site in red) to an open ring structure (active site in blue, triacylglyceride in spacefill) [8] (see Lipase lid morph for an animation of this transition). The lid opening is accompanied by a change in secondary structure from a mostly beta-extended confirmation to a structure where more than half the active site is formed from alpha helices [9].
Colipase Coenzyme
Lipase is activated by colipase, a coenzyme that binds to the C-terminal, non-catalytic domain of lipase. Colipase is a 10kDa protein that is secreted by the pancreas in an inactive form. It has five conserved disulfide bonds (shown in yellow) [10], and 2 surfaces- a hydrophilic surface (site of lipase C-terminal interaction- shown in blue) and a hydrophobic surface (contains multiple hydrophobic loops to bridge the lipid- shown in white)[11]. Trypsin will then activate colipase before the cofactor can interact with lipase.
Colipase must be present for activation of lipase and acts as a bridge between lipase and the lipid. When colipase binds, active lipase is stabilized for the hydrophobic interaction with triacylglycerides [12]. Without colipase present, the accumulation of amphiphiles at the oil/water interface in the duodenum would prevent pancreatic lipase from binding to its substrate. [13]. Colipase and lipase contacts are opposite of the active site on the C-terminal (contacts are regions of pink and yellow, with water molecules shown in darker blue). The enzymes are bound by polar interactions such as salt bridges, hydrophobic interactions and hydrogen bonds[14].
In the presence of colipase, the enzyme is activated which moves the N-terminal flap(shown in red, active site in green) 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.[15] The reorganization of the flap also induces a second conformational change that creates the oxyanion hole.[16]
Lipase Catalytic Mechanism
Lipase activation at the lipid-water interface of triacylglycerides, in the presence of colipase and bile salts, is known as interfacial activation. For the hydroloysis reaction to take place, colipase anchors lipase to the lipid-water membrane of the micelle which causes a surface change on lipase. Colipase's four hydrophobic loops interact with the hydrophobic atmosphere of the triacylglyceride. This initiates active site binding to the lipid, and lid opening to reveal a more hydrophobic environment for the triacylglycerol. This in turn, allows the triacylglycerol to interact with key active site residues like the catalytic triad. A diverse array of lipase enzymes can be found in nature. Though the different forms occupy diverse protein scaffolds, most are built upon an alpha/beta hydrolase fold[17][18] and possess a chymotrypsin-like catalytic triad comprised of an acidic residue, a histidine, and a serine nucleophile. In the case of horse pancreatic lipase, the catalytic triad is comprised of Ser 152, Asp 176 and His 263. [19]. This catalytic triad functions like most found in nature. First, aspartic acid forms a hydrogen bond with His 263, increasing the pKa of the histidine imidazole nitrogen. This allows the histidine to act as a powerful general base and deprotonate the serine. The deprotonated serine then can serve as a nucleophile and attack the ester carbonyl of one of the fatty acids on the 1 or 3 carbons of the glycerol backbone of the lipid substrate. Upon attacking the lipid, a negatively charged tetrahedral intermediate is formed (Reaction 1). It is stabilized in the oxyanion hole by two residues: Phe 77 and Leu 153.
A water molecule then donates a proton to the histidine, creating a reactive hydroxyl anion. The hydroxyl anion can then attack the carbonyl carbon of the lipid, forming another negatively charged tetrahedral intermediate which is stabilized in the oxyanion hole (Reaction 3).
Methoxyundecylphosphinic acid (MUP) (purple), a C11 alkyl phosphonate, is a competitive inhibitor of pancreatic lipase. It binds directly in the active site pocket. There are also five B-octylglucoside (gray and red) molecules which associate with lipase. MUP forms hydrogen bonds with four residues: 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 [20].
MUP was shown to be further stabilized by van der Waals contacts with hydrophobic side chains Ala 178, Phe 215, Pro l80, Tyr ll4, Leu 213 (shown in blue).
Protein - Substrate Interactions
Lipase binds substrates such as cholesteryl linoleate with numerous hydrophobic contacts. As is seen here, the lipase interacts with the alkyl group of cholesteryl linoleate via a hydrophobic rift within the protein. This rift orients the molecule to optimize the lipolysis reaction.
Shown in this scene is lipase from the yeast Candida rugosa in complex with two molecules of cholesteryl linoleate (grey). The active site residues including Ser152, Asp176, and His263 are shown in red stick representation. Lipase can accommodate two lipid molecules due to the fact that it's two identical subunits catalyze an identical reaction. One lipase molecule can catalyze two lipolysis reactions at a time.
Clinical Significance
Pancreatic lipase is secreted into the duodenum through the duct system of the pancreas. In a healthy individual, it is at very low concentration in serum. Under extreme disruption of pancreatic function, such as pancreatitis or pancreatic cancer, the pancreas may begin to digest itself and release pancreatic enzymes including pancreatic lipase into serum. Measurement of serum concentration of pancreatic lipase can therefore aid in diagnosis of acute pancreatitis.[21]. Due to lipase's activity in the digestion and absorption of fat, there has been a growing market for lipase inhibitors for weight loss pharmaceuticals. The most popular is Orlistat (or Xenical®) which is a natural product from Streptomyces toxytricini and is the hydrogenation product of lipostation- an irreversible lipase inhibitor. This inhibitor also acts by binding Ser152, producing an ester which hydrolyzes so slow that it is practically irreversible [22].
↑Thomas, A. etc. "Role of the Lid Hydrophobicity Pattern in Pancreatic Lipase Activity", The Journal of Biological Chemistry, 2005 September 22; 270 (48): 40074-40083.
↑"Colipase". Wikipedia: The Free Encyclopedia. 5 July 2011 [5]
↑Crandall,W., Lowe, M. "Colipase Residues Glu64 and Arg65 Are Essential for Normal Lipase-mediated Fat Digestion in the Presence of Bile Salt Micelles" Journal of Biological Chemistry, 2001, (276) 12505-12512
↑van Tilbeurgh H, etc."Structure of the pancreatic lipase-procolipase complex", 1992 Sep 10;359(6391):159-62. PMID:1522902.[6]
↑Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman I. Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science. 1991 Aug 23;253(5022):872-9. PMID:1678899
↑Ollis DL, Cheah E, Cygler M, Dijkstra B, Frolow F, Franken SM, Harel M, Remington SJ, Silman I, Schrag J, et al.. The alpha/beta hydrolase fold. Protein Eng. 1992 Apr;5(3):197-211. PMID:1409539
↑Bourne Y, Martinez C, Kerfelec B, Lombardo D, Chapus C, Cambillau C. Horse pancreatic lipase. The crystal structure refined at 2.3 A resolution. J Mol Biol. 1994 May 20;238(5):709-32. PMID:8182745 doi:https://dx.doi.org/10.1006/jmbi.1994.1331
