Lipase

spinqualitylabelsall models Structure of Pancreatic Lipase (PDB entry 1hpl) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction 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 . The reaction catalyzed by the enzyme is shown below. Further breakdown ultimately results in 2-monoacylglycerols and free fatty acids . 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 . 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 Molecular Playground/Pancreatic Lipase. Structure Pancreatic lipase is a 50 kDa protein, consisting of two identical, 449 residue chains . 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 . 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 . 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 . 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) . 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 . 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) , 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). 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 . Without colipase present, the accumulation of amphiphiles at the oil/water interface in the duodenum would prevent pancreatic lipase from binding to its substrate. . 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 . 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. The reorganization of the flap also induces a second conformational change that creates the oxyanion hole. 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 4 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 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. . 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. File:M0218.stg01.gif The carbonyl reforms with the glycerol backbone segment acting as the leaving group (Reaction 2). File:M0218.stg02.gif A water molecule then donates a proton to the histidine, creating a reactive hydroxyl anion, which can attack the carbonyl carbon of the lipid, forming another negatively charged tetrahedral intermediate which is stabilized in the oxyanion hole (Reaction 3). File:M0218.stg03.gif Upon reformation of the carbonyl, the catalytic serine is released and monoglyceride and fatty acid monomers diffuse away (Reaction 4). File:M0218.stg04.gif Inhibition of Pancreatic Lipase 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 . 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.. 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 .

3D Structures of Lipase

Update November 2011

Eukaryote natives:

Molecular Playground/Pancreatic Lipase – hLip – horse
chymotrypsin – hLip – human - gastric
3jw8, 3hju – mono-glyceride hLip
1jmy – hBSSL
1akn – cBSSL – cattle
2bce - cBSSL (mutant)
1f6w - cBSSL – catalytic domain
3o0d – Lip – Yarrowia lipolytica
1gpl – Lip – Guinea pig

Prokaryote natives:

3guu, 1lbs, 1lbt, 1tca, 1tcb, 1tcc – CaLipA – Candida antarctica
2veo – CaLipA – closed state
3icv – CaLipB (mutant)
1gz7, 1lpm, 1lps– CrLip 2 – Candida rugosa - closed state
1crl, 1trh – CrLip – open state
1llf – Lip – Candida cylindracea
3g7n – Lip - Penicillium expansum
1tia - Lip – Penicillium camemberti
2qua, 2qub – LipA – Serratia marcescens
2hih – Lip – Staphylococcus hyicus
2fx5 – Lip – Pseudomonas mendocina
1yzf – Lip – Enterococcus faecalis
1dt3, 1dt5, 1dte, 1du4, 1ein, 1tib – TlLip - Thermomyces lanuginose
1jfr – Lip – Streptomyces exfoliates
1oil – BcLip - Burkholderia cepacia
2lip – BcLip – open state
1cvl – Lip – Chromobacterium viscosum
1lgy – Lip II – Rhizopus niveus
1tic - Lip – Rhizopus oryzae
1thg – Lip – Geotrichum candidum
3tgl, 4tgl, 1tgl – RmLip– Rhyzomucor miehei
2zvd – PsLip - Pseudomonas sp. – open state
2z8x - PsLip – extracellular
2zj6, 2zj7 – PsLip (mutant)
2z8z – PsLip(mutant) – closed state
3lip, 3a6z - Lip - Pseudomonas cepacia – open state
1qge, 1tah – Lip – Pseudomonas glumae
2w22 – Lip – Geobacillus thermocatenulatus
1ji3, 1ku0 – Lip – Bacillus stearothermophilus
1ah7 - Lip – Bacillus cereus
2qxt, 2qxu, 1isp, 1i6w - BsLip – Bacillus subtilis
3d2a, 3d2b, 3d2c, 1t2n, 1t4m, 3qmm - BsLip (mutant)
2ory – Lip – Photobacterium lypoliticum
2z5g, 2dsn – Lip T1 – Geobacillus zalihae
3p94 – Lip – Parabacteroides distasonis
3ngm – Lip – Gibberella zeae

Lipase/colipase complexes. The colipase is a co-enzyme whose binding to lipase optimizes the enzymatic activity

1n8s – hLip+colipase II
1eth, 1lpa - Lip+colipase II - pig

Hormone-sensitive-lipases (LIPE) hydrolyze the first fatty acid of the triacylglycerol substrate

3k6k – EstE7(LIPE) – metagenome library
3fak, 3dnm – EstE5(LIPE) – metagenome library
1evq – AaEst2(LIPE) – Alicyclobacillus acidocaldarius
1u4n – AaEst2(LIPE) (mutant)

Putative lipases; Proteins with unknown function but structural similarity to lipase obtained in structural genomics projects.

2rau - Lip – Sulfolobus solfataricus
3bxp, 3d3n - Lip – Lactobacillus plantarum
3e0x - Lip – Clostridium acetobutylicum
1z8h – Lip – Nostoc sp. PCC 712
1vj3 - Lip – Nostoc sp.
3bzw – Lip - Bacteroides thetaiotaomicron
2pbl – Lip - Silicibacter

Lipase + inhibitors

3jwe, 3pe6 - mono-glyceride hLip + SAR629 – covalent inhibitor
3l1h – EstE5(LIPE)+FeCl3 – noninvasive inhibitor
3l1i, 3l1j - EstE5(LIPE)+CuSO4 – noninvasive inhibitor
3lij - EstE5(LIPE)+ZnSO4– noninvasive inhibitor
3h18, 3h17 - EstE5 (LIPE)+PMSF
3h19, 3h1b, 3h1a – EstE5 (LIPE)+methyl alcohol
3h1a – EstE5 SLIPE)+ethyl alcohol
3h19 – EstE5 SLIPE)+isopropyl alcohol
3g9t, 3g9u - EstE5 (HSLIPE)+p-nitrophenyl butyrate
3g9z - EstE5 (LIPE) +p-nitrophenyl caprylate
2nw6 – BcLip+ S inhibitor
4lip, 5lip, 1r4z, 1r50 – BcLip+ Rc-(Rp,Sp)-1,2-dioctylcarbamoyl-glycero-3-O-phosphonate
1r4z – BsLip+Rc-IPG-phosphonate
1r50 - BsLip+Sc-IPG-phosphonate
1k8q - Lip+phosphonate – dog
1ex9 – Lip+Rc-(Rp,Sp)-1,2-dioctylcarbamoyl-glycero-3-O-phosphonate – Pseudomonas aeruginosa
5tgl – RmLip+N-hexyl-phosphonate
1lpb – Lip (pig)+colipase+C11 alkyl phosphonate
3icw – CaLipB (mutant) +methyl hydrogen R hexylphosphonate
3a70 – PsLip+diethyl phosphate

Lipase conjugated with analogs to its reaction intermediates

1lpn, 1lpo, 1lpp – CrLip+ sulfonates
3rar – CrLip+ phosphonate
1qz3 – EaEst2(mutant) (LIPE)+hexadecanesulfonate

Lipase showing bile-salt binding site

1aql – cBSSL+taurocholate

Lipase with substrate bound at active site

2zyh – AfLip (mutant)+fatty acid – Archaeoglobus fulgidus
2zyi - AfLip+fatty acid+Ca
2zyr - AfLip+fatty acid+Mg
2zys - AfLip+fatty acid+Cl
1gt6 – TlLip+oleic acid - lipid ligand

Lipase conjugated to transition-state analogs showing the binding mode of the enzyme catalysis

1ys1 – BhLip+hexylphosphonic acid (R) 2-methyl-3-phenylpropyl ester
1ys2 – BhLip+hexylphosphonic acid (S) 2-methyl-3-phenylpropyl ester
1hqd – Lip+1-phenoxy-2-acrtoxy butane – Pseudomonas cepacia

Lipase+lipase chaperone

2es4 – Lip+lipase chaperone C-terminal - Burkholderia glumae

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