Sandbox Reserved 191

This Sandbox is Reserved from Feb 02, 2011, through Jul 31, 2011 for use by the Biochemistry II class at the Butler University at Indianapolis, IN USA taught by R. Jeremy Johnson. This reservation includes Sandbox Reserved 191 through Sandbox Reserved 200.

To get started: Click the edit this page tab at the top. Save the page after each step, then edit it again. Click the 3D button (when editing, above the wikitext box) to insert a 3D applet Jmol scene window. show the Scene authoring tools, create a molecular scene, and save it. Copy the green link into the page. Add a description of your scene. Use the buttons above the wikitext box for bold, italics, links, headlines, etc. More help: Help:Editing

spinqualitylabelsall models Palmitoyl-protein thioesterase 1 (PPT-1) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Introduction Palmitoyl-protein thioesterase 1 (PPT-1) is a small glycoprotein hydrolase found in the lysosome that breaks the thioester bond between cysteine amino acids and palmitic acid. PPT-1 is a homodimer that is composed primarily of alpha helices and beta sheets with a hydrophobic groove that allows the palmitic acid[1] to bind, exposing the thioester bond to the catalytic triad. PPT-1 was first found as an enzyme that removed palmitate from GTPase HRas, but over time it has been discovered to be able to able to remove palmitate from other proteins as well . When PPT-1 is not functioning properly, lipid modified proteins can build up in the cells, causing lysosomal storage diseases and aiding in tumor formation Surface view of PPT-1 showing the hydrophobic groove and palmitate Catalytic Triad PPT-1's structure creates an external hydrophobic groove that binds the palmitate acid in-between carbon 4 and 5. The acid binds in a gauche conformations [2] creating a kink in the acid chain. This bending could suggest that PPT-1 was originally designed to react with unsaturated fatty acid with cis-double bonds. The catalytic triad is composed of Serine 115, Aspartate 233, and Histidine 289. The Serine is "deprotonated" by the Histidine and attacks the carbonyl carbon of the palmitic acid. The negative charge is pushed onto the oxygen and is possibly stabilized by a water molecule. The tetrahedral intermediate collapses and kicks the palmatic acid off of the cysteine residue. File:Simple RXN.png The basic reaction Inhibitors of PPT-1 PPT-1 is a lysosomal enzyme which has a serine lipase consensus sequence; a key characteristic of lysosomal enzymes. Despite having a serine lipase consensus sequence, PPT-1, is not deactivated by phenylmethylsulfonyl fluoride (PMSF)[3], a common serine-modifying reagent. Hexadecylsulfonylfluoride (HDSF) is a serine-modifying reagent that is able to inhibit the actions of PPT-1 by binding to PPT-1. Unlike other inhibitors, HDSF is able to fit in the narrow, hydrophobic groove of PPT-1 leading away from the active site of PPT-1. PMSF is unable to fit into this small narrow groove due to steric constraints that relate to the unique structure of the substrate-binding site of PPT-1. The sulphur of HDSF will bind to SER-115 in the active site of PPT-1 via a sulfonylation reaction and thus will inhibit the actions of PPT-1. Diseases Associated With PPT-1 Mutations Associated with INCL Infantile Neuronal Ceroid Lipofuscinosis (INCL)[4] is a recessively inherited disease that is associated with a decrease in PPT-1 activity due to mutations of the PPT-1 enzyme. Onset of symptoms which include retinal blindness, ataxia, seizures, and cortical atrophy of the brain, begin 1-2 years after birth. Death typically occurs between the ages of 8-11. Several mutations in the 1p32 chromosome have been identified to cause INCL. Common Mutation associated with JNCL and LINCL involve mutations Far Away From Active Site Juvenile NCL (JNCL) and Late-infantile NCL (LINCL)are less severe forms of INCL in which onset of symptoms occur much later in life; between the ages of 30-40. The mutations associated with JNCL and LINCL occur away from the active site of PPT-1 resulting in a higher activity of the PPT-1 enzyme. A common mutation associated with JNCL and LINCL involves the mutation of Thr-75. Unfortunately not much is known on how to treat INCL. However, in both JNCL and LINCL, activity of PPT-1 has only a 2% activity rate compared to normal PPT-1 activity. This suggests that a small increase in activity of PPT-1 may aid in delaying the symptoms associated with INCL. One way in which to increase the activity of PPT-1 is to use protein chaperones [5] that help refold PPT-1 in the endoplasmic reticulm[6]. Although this is not a cure for INCL, by increasing the activity of PPT-1 the life expectancy for individuals with INCL can be greatly increased. Despite the life-threatening diseases associated with decreased PPT-1 activity there are cases in which PPT-1 activity is too high and thus needs to be inhibited. Proteins involved in signaling and growth are post-translationally modified. PPT-1 is involved with removing palmitate from lipid-modified proteins and this palmitoylation [7] is necessary for membrane association. When PPT-1 is overexpressed, cells become protected from cell death, thus there is an increase in the growth of cells which leads to tumor formation. Researchers have found that when PPT-1 was inhibited in a culture of tumor cells, the tumor cells died. Mutations in Palmitoyl Protein Thiosterase 1 Mutations in Palmitoyl Protein Thioesterase 1 (PPT-1) can cause three types of disorders: Infantile Neuronal Ceroid Lipofuscinosis (INCL) [8], Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) [9], and Juvenile Neuronal Cerioid Lipofuscinosis (JNCL)[10]. The severity of most of the mutations are dependent upon their location inside the protein with respect to the catalytic triad. “Mutations that affect catalysis or substrate binding or disrupt proper folding of the core result in inactive enzymes and lead to a severe clinical phenotype” . Other mutations that cause less severe disorders can sometimes retain some residual thioesterase activity. These less severe mutations are believed to have small, local changes in areas of the protein that are far away from the catalytic triad and palmitate binding site. A more detailed explanation of how some of the different disorders arise through mutations is explained below. Mutations leading to INCL Various mutations have been found in INCL patients . Most of these mutations are caused by nonsense or missense mutations within close proximity to the catalytic triad. These mutations lead to an inactive PPT-1 enzyme as they are predicted to create unfavorable steric, polar, and electrostatic interactions that could disturb the nucleophilic elbow. The nucleophilic elbow is responsible for proper location and orientation of the Ser-115. Catalytic ability would be greatly reduced if the original position of Ser-115 was altered because it must be properly orientated to be activated by His-289 in order to attack the substrate. One example of mutation such as Val181Met and Glu184Lys give a good depiction of how the increase in size in the mutated amino acids and positive charge on lysine mutation would create steric and polar clashes with the adjacent helices of the binding pocket compared to the Normal Val-181 & Glu-184 Normal Arg-122 which is described in more detail below. Known INCL Mutations 1. Arg122Trp 2. Phe225Ser 3. Gly118Asp 4. Gly42Gln 5. Tyr109Asp 6. His39Asp 7. Glu184Lys 8. Val181Met Mutations Associated with INCL Arg122Trp The most common mutation that occurs in PPT-1 is a single missense mutation of Arg122Trp. Arg-122 is located immediately after the nucleophilic elbow of PPT-1. The sidechain of Arg-122 has a main function to control the spacing between the αC and α6 helices in this region. This is done by Arg-122 having Three Hydrogen Bonds with three adjacent amino acids: Ile-205, Asn-206, and Gln-205. “Mutation of Arg-122 to Trp means not only a loss of those three hydrogen bonds but also a steric and polarity mismatch with the surrounding residues” . These Steric Clashes will cause a misfolding of the enzyme’s core and cause it to be trapped in the endoplasmic reticulum which results in no detectable PPT-1 activity. Mutation leading to LINCL LINCl is caused by a mutation in the CLN2 gene which “codes for a lysosomal pepstatin-insensitive acid protease that is deficient in LINCL patients” . Mutations that tend to lead to LINCL still disrupt the active site and binding pocket geometry but do not do it to the degree that is seen in INCL. One LINCL mutation is Gln177Glu. The Gln-177 acts as a hydrogen bond donor to Ala-171 and Ala-183 and as a hydrogen bond acceptor from Ile-200. It is presumed that the mutation to Glu-177 would cause a conformation change of the helices in the area. Ala-171 and Ala-183 make hydrophobic contact with the palmitate, and so the altered conformation associated with those two amino acids would decrease binding affinity of the palmitate, decreasing the catalytic activity but not to the extent of the mutations seen in INCL. Mutations leading to JNCL JNCL is caused by a mutation in the CLN3 gene which codes for a lysosomal membrane protein of unknown function . Unlike the mutations that cause INCL and LINCL, mutations that lead to JNCL are located away from the active site and are seen to cause less damage to the overall structure of PPT-1. Some of the mutations in JNCL have been noted as retaining a low level of PPT-1 activity as the catalytic site is left fairly unperturbed. Mutations associated with JNCl are found in two locations, Thr75Pro with Asp79Gly and Tyr247His with Gly250Val. These mutations are predicted to disturb the geometry of α1, increase the flexibility of the region, and alter the antiparallel βsheet motif in βa and βb compared to the Normal Tyr-247 & Gly-250.

External Resources

[1] Wikipedia page on Gauche Effect

[2] Wikipedia page on palmitic acid.

[3] Wikipedia page on Infantile neuronal ceroid lipofuscinosis

[4] Wikipedia page on PMSF

[5] Wikipedia page on Protein Chaperones

[6] Wikipedia page on Endoplasmic reticulum

[7] Wikipedia page on Palmitoylation