1exw: Difference between revisions

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CRYSTAL STRUCTURE OF PALMITOYL PROTEIN THIOESTERASE 1 COMPLEXED WITH HEXADECYLSULFONYL FLUORIDE

OverviewOverview

Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.

About this StructureAbout this Structure

1EXW is a Single protein structure of sequence from Bos taurus with and as ligands. Active as Palmitoyl-protein hydrolase, with EC number 3.1.2.22 Full crystallographic information is available from OCA.

ReferenceReference

Structural basis for the insensitivity of a serine enzyme (palmitoyl-protein thioesterase) to phenylmethylsulfonyl fluoride., Das AK, Bellizzi JJ 3rd, Tandel S, Biehl E, Clardy J, Hofmann SL, J Biol Chem. 2000 Aug 4;275(31):23847-51. PMID:10801859

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-[[Image:1exw.jpg|left|200px]]<br /><applet load="1exw" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1exw.jpg|left|200px]]<br /><applet load="1exw" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1exw, resolution 2.40&Aring;" />
 '''CRYSTAL STRUCTURE OF PALMITOYL PROTEIN THIOESTERASE 1 COMPLEXED WITH HEXADECYLSULFONYL FLUORIDE'''<br />
 ==Overview==
-Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal, enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine, residues in proteins. Deficiency in this enzyme results in a severe, neurodegenerative storage disorder, infantile neuronal ceroid, lipofuscinosis. Although the primary structure of PPT1 contains a serine, lipase consensus sequence, the enzyme is insensitive to commonly used, serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and, diisopropylfluorophosphate. In the current paper, we show that the active, site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner., The apparent K(i) of the inhibition was 125 micrometer (in the presence of, 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation, (k(2)) was 3.3/min, a value similar to previously described sulfonylation, reactions. PPT1 was crystallized after inactivation with HDSF, and the, structure of the inactive form was determined to 2.4 A resolution. The, hexadecylsulfonyl was found to modify serine 115 and to snake through a, narrow hydrophobic channel that would not accommodate an aromatic sulfonyl, fluoride. Therefore, the geometry of the active site accounts for the, reactivity of PPT1 with HDSF but not PMSF. These observations suggest a, structural explanation as to why certain serine lipases are resistant to, modification by commonly used serine-modifying reagents.
+Palmitoyl-protein thioesterase-1 (PPT1) is a newly described lysosomal enzyme that hydrolyzes long chain fatty acids from lipid-modified cysteine residues in proteins. Deficiency in this enzyme results in a severe neurodegenerative storage disorder, infantile neuronal ceroid lipofuscinosis. Although the primary structure of PPT1 contains a serine lipase consensus sequence, the enzyme is insensitive to commonly used serine-modifying reagents phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In the current paper, we show that the active site serine in PPT1 is modified by a substrate analog of PMSF, hexadecylsulfonylfluoride (HDSF) in a specific and site-directed manner. The apparent K(i) of the inhibition was 125 micrometer (in the presence of 1.5 mm Triton X-100), and the catalytic rate constant for sulfonylation (k(2)) was 3.3/min, a value similar to previously described sulfonylation reactions. PPT1 was crystallized after inactivation with HDSF, and the structure of the inactive form was determined to 2.4 A resolution. The hexadecylsulfonyl was found to modify serine 115 and to snake through a narrow hydrophobic channel that would not accommodate an aromatic sulfonyl fluoride. Therefore, the geometry of the active site accounts for the reactivity of PPT1 with HDSF but not PMSF. These observations suggest a structural explanation as to why certain serine lipases are resistant to modification by commonly used serine-modifying reagents.
 ==About this Structure==
-EXW is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with NAG and HDS as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Palmitoyl-protein_hydrolase Palmitoyl-protein hydrolase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.2.22 3.1.2.22] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1EXW OCA].
+EXW is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Bos_taurus Bos taurus] with <scene name='pdbligand=NAG:'>NAG</scene> and <scene name='pdbligand=HDS:'>HDS</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Palmitoyl-protein_hydrolase Palmitoyl-protein hydrolase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.2.22 3.1.2.22] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1EXW OCA].
 ==Reference==
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 [[Category: Single protein]]
 [[Category: Clardy, J.]]
-[[Category: III, J.J.Bellizzi.]]
+[[Category: III, J J.Bellizzi.]]
 [[Category: HDS]]
 [[Category: NAG]]
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 [[Category: pmsf]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 14:24:45 2007''
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