The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase
Identifieur interne : 001F08 ( Istex/Corpus ); précédent : 001F07; suivant : 001F09The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase
Auteurs : Wenjin Zheng ; Stephen Albert Johnston ; Leemor Joshua-TorSource :
- Cell [ 0092-8674 ] ; 1998.
English descriptors
- Teeft :
- Acta cryst, Active site, Active site cleft, Active site cleft region, Active site clefts, Active site cysteine, Active sites, Amide bond, Amino, Amino acid increments, Amino acids, Amino terminus, Aminopeptidase, Aminopeptidase activity, Bacterial bleomycin hydrolase, Biol, Bleomycin, Bleomycin hydrolase, Bleomycin hydrolysis, Bond angles, Bond lengths, Carboxylate, Carboxylate oxygens, Carboxypeptidase, Carboxypeptidase activity, Catalytic residues, Cell dimensions, Central channel, Cleavage, Cleavage site, Crystal structure, Cysteine, Cysteine protease, Cysteine proteinase, Data collection, Deletion, Deletion variants, Dihedral angles, Drop vapor diffusion method, Electron density, Electron density maps, Electrospray mass spectrometry, Endopeptidase, Endopeptidase activity, Equal amounts, Gal6, Gal6p, Gene fragment, Human bleomycin hydrolase, Hydrolase, Ionic interactions, Last shell, Leupeptin, Lysine, Mass spectrometry, Molecular mass, Molecular ruler, Mutant, Mutant protein, Mutant proteins, National institutes, National synchrotron light source, Nucleic acid binding activity, Nucleophilic cysteine, Other proteases, Oxyanion hole, Peptidase, Peptidase activity, Peptide, Peptide substrate, Positional specificity, Protease, Proteasome, Protein solution, Protein structures, Ramachandran plot, Research imaging plate detector system, Reservoir solution, Side chain, Single crystal, Substrate specificity, Terminus, Terminus controls, Tris buffer, Variant, Wild type, Zheng.
Abstract
Abstract: The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.
Url:
DOI: 10.1016/S0092-8674(00)81150-2
Links to Exploration step
ISTEX:FB9D51A6E87039034741F73FC940D84C74E5FE2FLe document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</title><author><name sortKey="Zheng, Wenjin" sort="Zheng, Wenjin" uniqKey="Zheng W" first="Wenjin" last="Zheng">Wenjin Zheng</name><affiliation><mods:affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</mods:affiliation></affiliation></author><author><name sortKey="Johnston, Stephen Albert" sort="Johnston, Stephen Albert" uniqKey="Johnston S" first="Stephen Albert" last="Johnston">Stephen Albert Johnston</name><affiliation><mods:affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</mods:affiliation></affiliation></author><author><name sortKey="Joshua Tor, Leemor" sort="Joshua Tor, Leemor" uniqKey="Joshua Tor L" first="Leemor" last="Joshua-Tor">Leemor Joshua-Tor</name><affiliation><mods:affiliation>E-mail: leemor@cshl.org</mods:affiliation></affiliation><affiliation><mods:affiliation>W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:FB9D51A6E87039034741F73FC940D84C74E5FE2F</idno><date when="1998" year="1998">1998</date><idno type="doi">10.1016/S0092-8674(00)81150-2</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-7RX1W659-8/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001F08</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001F08</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</title><author><name sortKey="Zheng, Wenjin" sort="Zheng, Wenjin" uniqKey="Zheng W" first="Wenjin" last="Zheng">Wenjin Zheng</name><affiliation><mods:affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</mods:affiliation></affiliation></author><author><name sortKey="Johnston, Stephen Albert" sort="Johnston, Stephen Albert" uniqKey="Johnston S" first="Stephen Albert" last="Johnston">Stephen Albert Johnston</name><affiliation><mods:affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</mods:affiliation></affiliation></author><author><name sortKey="Joshua Tor, Leemor" sort="Joshua Tor, Leemor" uniqKey="Joshua Tor L" first="Leemor" last="Joshua-Tor">Leemor Joshua-Tor</name><affiliation><mods:affiliation>E-mail: leemor@cshl.org</mods:affiliation></affiliation><affiliation><mods:affiliation>W. 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Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Cell</title><title level="j" type="abbrev">CELL</title><idno type="ISSN">0092-8674</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998">1998</date><biblScope unit="volume">93</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="103">103</biblScope><biblScope unit="page" to="109">109</biblScope></imprint><idno type="ISSN">0092-8674</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0092-8674</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Acta cryst</term><term>Active site</term><term>Active site cleft</term><term>Active site cleft region</term><term>Active site clefts</term><term>Active site cysteine</term><term>Active sites</term><term>Amide bond</term><term>Amino</term><term>Amino acid increments</term><term>Amino acids</term><term>Amino terminus</term><term>Aminopeptidase</term><term>Aminopeptidase activity</term><term>Bacterial bleomycin hydrolase</term><term>Biol</term><term>Bleomycin</term><term>Bleomycin hydrolase</term><term>Bleomycin hydrolysis</term><term>Bond angles</term><term>Bond lengths</term><term>Carboxylate</term><term>Carboxylate oxygens</term><term>Carboxypeptidase</term><term>Carboxypeptidase activity</term><term>Catalytic residues</term><term>Cell dimensions</term><term>Central channel</term><term>Cleavage</term><term>Cleavage site</term><term>Crystal structure</term><term>Cysteine</term><term>Cysteine protease</term><term>Cysteine proteinase</term><term>Data collection</term><term>Deletion</term><term>Deletion variants</term><term>Dihedral angles</term><term>Drop vapor diffusion method</term><term>Electron density</term><term>Electron density maps</term><term>Electrospray mass spectrometry</term><term>Endopeptidase</term><term>Endopeptidase activity</term><term>Equal amounts</term><term>Gal6</term><term>Gal6p</term><term>Gene fragment</term><term>Human bleomycin hydrolase</term><term>Hydrolase</term><term>Ionic interactions</term><term>Last shell</term><term>Leupeptin</term><term>Lysine</term><term>Mass spectrometry</term><term>Molecular mass</term><term>Molecular ruler</term><term>Mutant</term><term>Mutant protein</term><term>Mutant proteins</term><term>National institutes</term><term>National synchrotron light source</term><term>Nucleic acid binding activity</term><term>Nucleophilic cysteine</term><term>Other proteases</term><term>Oxyanion hole</term><term>Peptidase</term><term>Peptidase activity</term><term>Peptide</term><term>Peptide substrate</term><term>Positional specificity</term><term>Protease</term><term>Proteasome</term><term>Protein solution</term><term>Protein structures</term><term>Ramachandran plot</term><term>Research imaging plate detector system</term><term>Reservoir solution</term><term>Side chain</term><term>Single crystal</term><term>Substrate specificity</term><term>Terminus</term><term>Terminus controls</term><term>Tris buffer</term><term>Variant</term><term>Wild type</term><term>Zheng</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>gal6p</json:string><json:string>bleomycin</json:string><json:string>gal6</json:string><json:string>cysteine</json:string><json:string>deletion</json:string><json:string>hydrolase</json:string><json:string>aminopeptidase</json:string><json:string>active site</json:string><json:string>terminus</json:string><json:string>leupeptin</json:string><json:string>carboxylate</json:string><json:string>peptide</json:string><json:string>peptidase</json:string><json:string>zheng</json:string><json:string>protease</json:string><json:string>substrate specificity</json:string><json:string>carboxypeptidase</json:string><json:string>mutant</json:string><json:string>proteasome</json:string><json:string>lysine</json:string><json:string>endopeptidase</json:string><json:string>biol</json:string><json:string>peptidase activity</json:string><json:string>amino</json:string><json:string>crystal structure</json:string><json:string>bleomycin hydrolase</json:string><json:string>last shell</json:string><json:string>terminus controls</json:string><json:string>peptide substrate</json:string><json:string>amino acids</json:string><json:string>mutant proteins</json:string><json:string>cleavage site</json:string><json:string>carboxypeptidase activity</json:string><json:string>side chain</json:string><json:string>active sites</json:string><json:string>active site cysteine</json:string><json:string>data collection</json:string><json:string>cleavage</json:string><json:string>variant</json:string><json:string>catalytic residues</json:string><json:string>wild type</json:string><json:string>active site clefts</json:string><json:string>active site cleft</json:string><json:string>carboxylate oxygens</json:string><json:string>molecular mass</json:string><json:string>nucleophilic cysteine</json:string><json:string>molecular ruler</json:string><json:string>endopeptidase activity</json:string><json:string>ionic interactions</json:string><json:string>oxyanion hole</json:string><json:string>deletion variants</json:string><json:string>mass spectrometry</json:string><json:string>aminopeptidase activity</json:string><json:string>nucleic acid binding activity</json:string><json:string>positional specificity</json:string><json:string>amino acid increments</json:string><json:string>bleomycin hydrolysis</json:string><json:string>amino terminus</json:string><json:string>amide bond</json:string><json:string>bacterial bleomycin hydrolase</json:string><json:string>other proteases</json:string><json:string>gene fragment</json:string><json:string>drop vapor diffusion method</json:string><json:string>equal amounts</json:string><json:string>protein solution</json:string><json:string>tris buffer</json:string><json:string>reservoir solution</json:string><json:string>cell dimensions</json:string><json:string>mutant protein</json:string><json:string>cysteine protease</json:string><json:string>single crystal</json:string><json:string>national synchrotron light source</json:string><json:string>research imaging plate detector system</json:string><json:string>central channel</json:string><json:string>electron density</json:string><json:string>bond lengths</json:string><json:string>bond angles</json:string><json:string>dihedral angles</json:string><json:string>ramachandran plot</json:string><json:string>electron density maps</json:string><json:string>national institutes</json:string><json:string>human bleomycin hydrolase</json:string><json:string>active site cleft region</json:string><json:string>cysteine proteinase</json:string><json:string>acta cryst</json:string><json:string>protein structures</json:string><json:string>electrospray mass spectrometry</json:string></teeft></keywords><author><json:item><name>Wenjin Zheng</name><affiliations><json:string>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</json:string></affiliations></json:item><json:item><name>Stephen Albert Johnston</name><affiliations><json:string>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</json:string></affiliations></json:item><json:item><name>Leemor Joshua-Tor</name><affiliations><json:string>E-mail: leemor@cshl.org</json:string><json:string>W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA</json:string></affiliations></json:item></author><arkIstex>ark:/67375/6H6-7RX1W659-8</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.</abstract><qualityIndicators><score>8.344</score><pdfWordCount>5386</pdfWordCount><pdfCharCount>31966</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>7</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>112</abstractWordCount><abstractCharCount>675</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</title><pmid><json:string>9546396</json:string></pmid><pii><json:string>S0092-8674(00)81150-2</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Cell</title><language><json:string>unknown</json:string></language><publicationDate>1998</publicationDate><issn><json:string>0092-8674</json:string></issn><pii><json:string>S0092-8674(00)X0119-5</json:string></pii><volume>93</volume><issue>1</issue><pages><first>103</first><last>109</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1998</json:string><json:string>20S</json:string></date><geogName></geogName><orgName><json:string>Keck Structural Biology Laboratory Cold Spring Harbor Laboratory Cold Spring Harbor, New York</json:string><json:string>National Synchrotron Light Source</json:string><json:string>Brookhaven National Laboratories</json:string><json:string>Bachem</json:string><json:string>Tobacco Research Council</json:string><json:string>National Synchroton Light Source</json:string><json:string>Mabel Beckman Foundation, the Robertson Research Fund, and the Cold Spring Harbor Association Fellowship</json:string><json:string>and National Institutes of Health</json:string><json:string>University of Texas-Southwestern</json:string><json:string>Texas-Southwestern Medical Center Dallas</json:string><json:string>Brookhaven National Laboratory</json:string><json:string>Johnston</json:string><json:string>CA</json:string><json:string>Joshua-Tor</json:string><json:string>National Institutes of Health</json:string></orgName><orgName_funder><json:string>Johnston</json:string><json:string>CA</json:string><json:string>Joshua-Tor</json:string><json:string>National Institutes of Health</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Clive Slaughter</json:string><json:string>L. J. Received</json:string><json:string>Winship Herr</json:string><json:string>David Corey</json:string><json:string>C. Access</json:string><json:string>Julie Rosenbaum</json:string><json:string>Lonny Berman</json:string><json:string>Meg Phillips</json:string><json:string>Malcolm Capel</json:string><json:string>Felix Frolow</json:string></persName><placeName><json:string>Specificity</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Larsen and Finley, 1997</json:string><json:string>Brunger, 1988</json:string><json:string>Enenkel and Wolf, 1993</json:string><json:string>Zheng and Johnston, 1997</json:string><json:string>Magdolen et al., 1993</json:string><json:string>Akiyama et al., 1981</json:string><json:string>Bromme et al., 1996</json:string><json:string>Otwinsowski, 1993</json:string><json:string>Klein et al., 1997</json:string><json:string>Adachi et al., 1997</json:string><json:string>Takeda et al., 1996</json:string><json:string>JoshuaTor et al., 1995</json:string><json:string>Schroder et al., 1993</json:string><json:string>Ferrando et al., 1996</json:string><json:string>Schechter and Berger, 1968</json:string><json:string>Tong et al., 1993</json:string><json:string>Zheng et al., 1997</json:string><json:string>Laskowski et al., 1993</json:string><json:string>Seemuller et al., 1996</json:string><json:string>Bacon and Anderson, 1988</json:string><json:string>Joshua-Tor et al., 1995</json:string><json:string>Lazo and Humphreys, 1983</json:string><json:string>Merritt and Murphy, 1994</json:string><json:string>Lowe et al., 1995</json:string><json:string>Chen and Hochstrasser, 1996</json:string><json:string>Kambouris et al., 1992</json:string><json:string>Xu and Johnston, 1994</json:string><json:string>Kraulis, 1991</json:string><json:string>Jones et al., 1991</json:string><json:string>Lupas et al., 1997</json:string><json:string>Lazo and Chabner, 1996</json:string><json:string>Mata et al., 1997</json:string><json:string>Chapot et al., 1993</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-7RX1W659-8</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - cell biology</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - developmental biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - General Biochemistry, Genetics and Molecular Biology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>1998</publicationDate><copyrightDate>1998</copyrightDate><doi><json:string>10.1016/S0092-8674(00)81150-2</json:string></doi><id>FB9D51A6E87039034741F73FC940D84C74E5FE2F</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-7RX1W659-8/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-7RX1W659-8/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-7RX1W659-8/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1998 Cell Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1998</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Section title: Article</note><note type="content">Figure 1: The Structure of the Gal6p Hexamer and Access to the Active Site (Top) A space-filling representation of the molecule shown as a side view with the six subunits shown in different colors. This figure as well as Figure 2A and Figure 2B were drawn with the program Molscript (Kraulis 1991) and rendered with RASTER3D (Bacon and Anderson 1988; Merritt and Murphy 1994). The protein is shaped as a cylinder with a channel through its middle. The channel is approximately 20 Å in diameter at the two rims and opens to a larger central cavity. The C terminus of each subunit folds back into the active site cleft. (Bottom) A slice through the middle of the protein along it's 3-fold axis. Four out of the six active sites are shown as distorted stars and the C terminus as a squiggle line labeled at the end with the letter C. Access to the active sites is through the central channel.</note><note type="content">Figure 2: Positioning of the C-Terminal Arm of Gal6 in the Active Site Clefts in the Wild-Type and Mutant Proteins (A) A superposition of the C-terminal arm and active site residues of the C73A variant (orange) on the active site cleft region of wild-type Gal6p (blue). The two C-terminal arms superimpose closely with K454 in the variant clearly seen in red as a well ordered residue occupying the S1 site and extending from the rest of the chain toward the catalytic residues. (B) A similar superposition of the active site cleft region of C73A/ΔK454 (dark orange) superimposed on the wild-type protein (blue). A453 extends further toward the catalytic residues in this variant compared to wild type and is occupying the S1 site. The glycine bulge of the wild-type protein switches to an extended conformation in the variant (yellow). (C) Cartoon illustrations of the active site structures of the wild type and the variants of Gal6p. Note that the C-terminal residues are color-coded in this and following cartoons. The scissors represent the active site cysteine and histidine at the cleavage site. In the Gal6 cartoon, the uncolored ovals represent the residues from the P1, P1', and P2' position of a peptide substrate.</note><note type="content">Figure 3: G450A Has a Different Processing Pattern from the Wild-Type Protein An electrospray mass spectrometry shows G450A protein cleaving either one or three residues of the C terminus.</note><note type="content">Figure 4: Mass Spectrometry Shows that Deletion of the C-Terminal Residues of Gal6p Changes Its Substrate Specificity A 12 mer peptide was used as the substrate. The peptide sequence and the cleavage sites are indicated at the top of the picture (arrows). The mass of the 12 mer has been framed. (A) Gal6p. (B) Deletion of C-terminal two residues Gal6Δ2. (C) Deletion of C-terminal three residues Gal6Δ3. (D) Deletion of C-terminal four residues Gal6Δ4. In (A), (B), and (C), the peaks labeled by stars are ligation products resulting in an increase of mass over that of the original substrate.</note><note type="content">Figure 5: Deletion of the C Terminus of Gal6p Results in an Increase of Its Sensitivity to Leupeptin (Top) Relative inhibition of wild-type and Gal6Δ3p by leupeptin on Arg-AMC and Ala-Arg-AMC substrates, respectively. (Bottom) A cartoon illustrating the rationale for the increased sensitivity of deletions of the C terminus to leupeptin by opening subsites previously occupied by the C-terminal residues.</note><note type="content">Figure 6: Possible Mechanism of Bleomycin Hydrolysis by Bleomycin Hydrolase</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</title><author xml:id="author-0000"><persName><forename type="first">Wenjin</forename><surname>Zheng</surname></persName><affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Stephen Albert</forename><surname>Johnston</surname></persName><affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Leemor</forename><surname>Joshua-Tor</surname></persName><email>leemor@cshl.org</email><note type="correspondence"><p>Correspondence: Leemor Joshua-Tor, 516-367-8821 (phone), 516-367-8873 (fax)</p></note><affiliation>To whom correspondence should be addressed.</affiliation><affiliation>W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA</affiliation></author><idno type="istex">FB9D51A6E87039034741F73FC940D84C74E5FE2F</idno><idno type="ark">ark:/67375/6H6-7RX1W659-8</idno><idno type="DOI">10.1016/S0092-8674(00)81150-2</idno><idno type="PII">S0092-8674(00)81150-2</idno></analytic><monogr><title level="j">Cell</title><title level="j" type="abbrev">CELL</title><idno type="pISSN">0092-8674</idno><idno type="PII">S0092-8674(00)X0119-5</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998"></date><biblScope unit="volume">93</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="103">103</biblScope><biblScope unit="page" to="109">109</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1998</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.</p></abstract></profileDesc><revisionDesc><change when="1998-02-25">Modified</change><change when="1998">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-7RX1W659-8/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>CELL</jid><aid>1129</aid><ce:pii>S0092-8674(00)81150-2</ce:pii><ce:doi>10.1016/S0092-8674(00)81150-2</ce:doi><ce:copyright type="other" year="1998">Cell Press</ce:copyright></item-info><head><ce:dochead><ce:textfn>Article</ce:textfn></ce:dochead><ce:title>The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</ce:title><ce:author-group><ce:author><ce:given-name>Wenjin</ce:given-name><ce:surname>Zheng</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Stephen Albert</ce:given-name><ce:surname>Johnston</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Leemor</ce:given-name><ce:surname>Joshua-Tor</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref><ce:cross-ref refid="FN1">‡</ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>leemor@cshl.org</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Correspondence: Leemor Joshua-Tor, 516-367-8821 (phone), 516-367-8873 (fax)</ce:text></ce:correspondence></ce:author-group><ce:date-received day="30" month="1" year="1998"></ce:date-received><ce:date-revised day="25" month="2" year="1998"></ce:date-revised><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>The Unusual Active Site of Gal6/Bleomycin Hydrolase Can Act as a Carboxypeptidase, Aminopeptidase, and Peptide Ligase</title></titleInfo><name type="personal"><namePart type="given">Wenjin</namePart><namePart type="family">Zheng</namePart><affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Stephen Albert</namePart><namePart type="family">Johnston</namePart><affiliation>Center for Biomedical Inventions, Departments of Medicine and Biochemistry, University of Texas-Southwestern Medical Center, Dallas, Texas 75235-8573, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Leemor</namePart><namePart type="family">Joshua-Tor</namePart><affiliation>E-mail: leemor@cshl.org</affiliation><affiliation>W. M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA</affiliation><description>Correspondence: Leemor Joshua-Tor, 516-367-8821 (phone), 516-367-8873 (fax)</description><description>To whom correspondence should be addressed.</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued><dateModified encoding="w3cdtf">1998-02-25</dateModified><copyrightDate encoding="w3cdtf">1998</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: The Gal6 protease is in a class of cysteine peptidases identified by their ability to inactivate the anti-cancer drug bleomycin. The protein forms a barrel structure with the active sites embedded in a channel as in the proteasome. In Gal6 the C termini lie in the active site clefts. We show that Gal6 acts as a carboxypeptidase on its C terminus to convert itself to an aminopeptidase and peptide ligase. The substrate specificity of the peptidase activity is determined by the position of the C terminus of Gal6 rather than the sequence of the substrate. We propose a model to explain these diverse activities and Gal6's singular ability to inactivate bleomycin.</abstract><note type="content">Section title: Article</note><note type="content">Figure 1: The Structure of the Gal6p Hexamer and Access to the Active Site (Top) A space-filling representation of the molecule shown as a side view with the six subunits shown in different colors. This figure as well as Figure 2A and Figure 2B were drawn with the program Molscript (Kraulis 1991) and rendered with RASTER3D (Bacon and Anderson 1988; Merritt and Murphy 1994). The protein is shaped as a cylinder with a channel through its middle. The channel is approximately 20 Å in diameter at the two rims and opens to a larger central cavity. The C terminus of each subunit folds back into the active site cleft. (Bottom) A slice through the middle of the protein along it's 3-fold axis. Four out of the six active sites are shown as distorted stars and the C terminus as a squiggle line labeled at the end with the letter C. Access to the active sites is through the central channel.</note><note type="content">Figure 2: Positioning of the C-Terminal Arm of Gal6 in the Active Site Clefts in the Wild-Type and Mutant Proteins (A) A superposition of the C-terminal arm and active site residues of the C73A variant (orange) on the active site cleft region of wild-type Gal6p (blue). The two C-terminal arms superimpose closely with K454 in the variant clearly seen in red as a well ordered residue occupying the S1 site and extending from the rest of the chain toward the catalytic residues. (B) A similar superposition of the active site cleft region of C73A/ΔK454 (dark orange) superimposed on the wild-type protein (blue). A453 extends further toward the catalytic residues in this variant compared to wild type and is occupying the S1 site. The glycine bulge of the wild-type protein switches to an extended conformation in the variant (yellow). (C) Cartoon illustrations of the active site structures of the wild type and the variants of Gal6p. Note that the C-terminal residues are color-coded in this and following cartoons. The scissors represent the active site cysteine and histidine at the cleavage site. In the Gal6 cartoon, the uncolored ovals represent the residues from the P1, P1', and P2' position of a peptide substrate.</note><note type="content">Figure 3: G450A Has a Different Processing Pattern from the Wild-Type Protein An electrospray mass spectrometry shows G450A protein cleaving either one or three residues of the C terminus.</note><note type="content">Figure 4: Mass Spectrometry Shows that Deletion of the C-Terminal Residues of Gal6p Changes Its Substrate Specificity A 12 mer peptide was used as the substrate. The peptide sequence and the cleavage sites are indicated at the top of the picture (arrows). The mass of the 12 mer has been framed. (A) Gal6p. (B) Deletion of C-terminal two residues Gal6Δ2. (C) Deletion of C-terminal three residues Gal6Δ3. (D) Deletion of C-terminal four residues Gal6Δ4. In (A), (B), and (C), the peaks labeled by stars are ligation products resulting in an increase of mass over that of the original substrate.</note><note type="content">Figure 5: Deletion of the C Terminus of Gal6p Results in an Increase of Its Sensitivity to Leupeptin (Top) Relative inhibition of wild-type and Gal6Δ3p by leupeptin on Arg-AMC and Ala-Arg-AMC substrates, respectively. (Bottom) A cartoon illustrating the rationale for the increased sensitivity of deletions of the C terminus to leupeptin by opening subsites previously occupied by the C-terminal residues.</note><note type="content">Figure 6: Possible Mechanism of Bleomycin Hydrolysis by Bleomycin Hydrolase</note><relatedItem type="host"><titleInfo><title>Cell</title></titleInfo><titleInfo type="abbreviated"><title>CELL</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued></originInfo><identifier type="ISSN">0092-8674</identifier><identifier type="PII">S0092-8674(00)X0119-5</identifier><part><date>1998</date><detail type="volume"><number>93</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>148</end></extent><extent unit="pages"><start>103</start><end>109</end></extent></part></relatedItem><identifier type="istex">FB9D51A6E87039034741F73FC940D84C74E5FE2F</identifier><identifier type="ark">ark:/67375/6H6-7RX1W659-8</identifier><identifier type="DOI">10.1016/S0092-8674(00)81150-2</identifier><identifier type="PII">S0092-8674(00)81150-2</identifier><accessCondition type="use and reproduction" contentType="copyright">©1998 Cell Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Cell Press, ©1998</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-7RX1W659-8/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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