Sandbox Reserved 191: Difference between revisions
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The binding surface of PPT-1 creates an external hydrophobic groove that binds the palmitate acid in-between carbon 4 and 5 (Figure 1). The acid binds in a [https://en.wikipedia.org/wiki/Gauche_effect gauche conformation] creating a <scene name='58/580839/Kink_in_acid/1'>kink </scene> | The binding surface of PPT-1 creates an external hydrophobic groove that binds the palmitate acid in-between carbon 4 and 5 (Figure 1). The acid binds in a [https://en.wikipedia.org/wiki/Gauche_effect gauche conformation] creating a <scene name='58/580839/Kink_in_acid/1'>kink </scene> | ||
in the acid chain. This bending suggests that PPT-1 was originally designed to react with an unsaturated fatty acid with cis-double bonds. The catalytic <scene name='43/436866/Triad_w_zoom_no_backbones/1'>triad</scene> is composed of Serine-115, Aspartate-233, and Histidine-289 <ref name="mutations" />. Ser-115 is deprotonated by His-289 and attacks the carbonyl carbon of the thioester bond connecting palmitic acid to the protein substrate. The negative charge is pushed onto the oxygen and is stabilized | in the acid chain. This bending suggests that PPT-1 was originally designed to react with an unsaturated fatty acid with cis-double bonds. The catalytic <scene name='43/436866/Triad_w_zoom_no_backbones/1'>triad</scene> is composed of Serine-115, Aspartate-233, and Histidine-289 <ref name="mutations" />. Ser-115 is deprotonated by His-289 and attacks the carbonyl carbon of the thioester bond connecting palmitic acid to the protein substrate. The negative charge is pushed onto the oxygen and is stabilized in an oxyanion hole by a water molecule. The tetrahedral intermediate collapses and kicks the palmatic acid off of the cysteine residue<ref name="mutations" />. | ||
[[Image:Rxn ppt1.jpg|400px|right|thumb|Figure 2: The basic enzymatic reaction catalyzed by PPT-1. Ser-115, depronated by His-289, attacks the carbonyl carbon of the thioester bond ]] | [[Image:Rxn ppt1.jpg|400px|right|thumb|Figure 2: The basic enzymatic reaction catalyzed by PPT-1. Ser-115, depronated by His-289, attacks the carbonyl carbon of the thioester bond ]] | ||
=Inhibitors of PPT-1= | =Inhibitors of PPT-1= | ||
'''<scene name='43/436866/Overall-3-rainbow/1'>PPT-1</scene>''' 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 [https://en.wikipedia.org/wiki/PMSF (PMSF)], a common serine-modifying reagent. <scene name='58/580839/Hdsf_by_itself/1'>Hexadecylsulfonylfluoride</scene> (HDSF) is a serine-modifying reagent that is able to <scene name='58/580839/Basic-hdsf-nosurface/4'> bind and inhibit PPT-1</scene>. Unlike other inhibitors, <scene name='58/580839/Basic-hdsf-surfacelook/2'>HDSF is able to fit in the narrow, hydrophobic groove of PPT-1</scene> 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 <scene name='58/580839/Hdsf_bound_to_ser-115-best/1'>bind to SER-115 in the active site of PPT-1</scene> via a | '''<scene name='43/436866/Overall-3-rainbow/1'>PPT-1</scene>''' 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 [https://en.wikipedia.org/wiki/PMSF (PMSF)], a common serine-modifying reagent. <scene name='58/580839/Hdsf_by_itself/1'>Hexadecylsulfonylfluoride</scene> (HDSF) is a serine-modifying reagent that is able to <scene name='58/580839/Basic-hdsf-nosurface/4'> bind and inhibit PPT-1</scene>. Unlike other inhibitors, <scene name='58/580839/Basic-hdsf-surfacelook/2'>HDSF is able to fit in the narrow, hydrophobic groove of PPT-1</scene> 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 <scene name='58/580839/Hdsf_bound_to_ser-115-best/1'>bind to SER-115 in the active site of PPT-1</scene> via a sulphonylation reaction and thus will inhibit the actions of PPT-1<ref name="INCL">PMID:10801859</ref>. | ||
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===Mutations leading to Infantile Neuronal Ceroid Lipofuscinosis=== | ===Mutations leading to Infantile Neuronal Ceroid Lipofuscinosis=== | ||
Various mutations have been found in INCL patients<ref name="Ryan-1">PMID:10191107</ref>. Most of these mutations are caused by nonsense or missense mutations within close proximity to the catalytic <scene name='43/436866/Triad_w_zoom_no_backbones/1'>triad</scene>. 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 <ref name="mutations" />. The nucleophilic elbow is responsible for proper location and orientation of the Ser-115. The catalytic activity of PPT-1 is greatly reduced if the positioning of Ser-115 is altered, as Ser-115 must be properly orientated to be activated by His-289 to be positioned to attack the substrate. An example of a INCL mutation such as <scene name='58/580837/Methionine/9'>Val181Met</scene> and <scene name='58/580837/Lysine_mutation/4'>Glu184Lys</scene> gives a good depiction of how the increase in size in the mutated amino acids and positive charge on the inserted lysine residue would create steric and polar clashes with the adjacent helices of the binding pocket compared to the <scene name='58/580837/Val181glu184/3'>normal Val-181 and Glu-184</scene>. <scene name='58/580837/Arginine_fine/5'> | Various mutations have been found in INCL patients<ref name="Ryan-1">PMID:10191107</ref>. Most of these mutations are caused by nonsense or missense mutations within close proximity to the catalytic <scene name='43/436866/Triad_w_zoom_no_backbones/1'>triad</scene>. 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 <ref name="mutations" />. The nucleophilic elbow is responsible for proper location and orientation of the Ser-115. The catalytic activity of PPT-1 is greatly reduced if the positioning of Ser-115 is altered, as Ser-115 must be properly orientated to be activated by His-289 to be positioned to attack the substrate. An example of a INCL mutation such as <scene name='58/580837/Methionine/9'>Val181Met</scene> and <scene name='58/580837/Lysine_mutation/4'>Glu184Lys</scene> gives a good depiction of how the increase in size in the mutated amino acids and positive charge on the inserted lysine residue would create steric and polar clashes with the adjacent helices of the binding pocket compared to the <scene name='58/580837/Val181glu184/3'>normal Val-181 and Glu-184</scene>. <scene name='58/580837/Arginine_fine/5'>Normal Arg-122</scene> which is below <ref name="Ryan-1">PMID:10191107</ref>. | ||
(*All mutation scenes are the proposed best rotamers of the mutation. These mutations were created through the use of the mutagenesis wizard in the PyMOL program) | (*All mutation scenes are the proposed best rotamers of the mutation. These mutations were created through the use of the mutagenesis wizard in the PyMOL program) | ||