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Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis
Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent. The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the modification of a single amino acid residue. Peptic di...
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| Published in: | The Journal of biological chemistry 1990-07, Vol.265 (19), p.10817-10820 |
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| Main Authors: | , |
| Format: | Article |
| Language: | English |
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| cites | cdi_FETCH-LOGICAL-c526t-41bf7fc662ef212d043c16f0eafe440720aeb4d2a6b976ed794bb8598dbc59993 |
| container_end_page | 10820 |
| container_issue | 19 |
| container_start_page | 10817 |
| container_title | The Journal of biological chemistry |
| container_volume | 265 |
| creator | Reddy, V.A. (New York State Department of Health, Albany, NY) Maley, F |
| description | Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent.
The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the
modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column
chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these
peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing
it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for
Asp-23 in the catalytic process. |
| doi_str_mv | 10.1016/s0021-9258(19)38518-7 |
| format | article |
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The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the
modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column
chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these
peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing
it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for
Asp-23 in the catalytic process.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/s0021-9258(19)38518-7</identifier><identifier>PMID: 2113524</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: American Society for Biochemistry and Molecular Biology</publisher><subject>Acetylglucosaminidase ; ACIDE ASPARTIQUE ; ACIDO ASPARTICO ; actine site amino acid ; affinity labelling ; Affinity Labels ; amino acid residue ; Amino Acid Sequence ; ASPARTIC ACID ; Aspartic Acid - genetics ; Base Sequence ; beta -fructofuranosidase ; BINDING SITE ; Binding Sites ; Catalysis ; Chromatography, High Pressure Liquid ; Exact sciences and technology ; FRUCTOFURANOSIDASA ; FRUCTOFURANOSIDASE ; Glycoside Hydrolases - antagonists & inhibitors ; Glycoside Hydrolases - genetics ; Glycoside Hydrolases - metabolism ; Glycosylation ; Inositol - analogs & derivatives ; Inositol - pharmacology ; Kinetics ; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase ; Mathematical analysis ; Mathematics ; Molecular Sequence Data ; Mutation ; Peptide Fragments ; Potential theory ; SACCHAROMYCES CEREVISIAE ; Saccharomyces cerevisiae - enzymology ; Saccharomyces cerevisiae - genetics ; Sciences and techniques of general use ; site-directed mutagenesis ; Trypsin</subject><ispartof>The Journal of biological chemistry, 1990-07, Vol.265 (19), p.10817-10820</ispartof><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-41bf7fc662ef212d043c16f0eafe440720aeb4d2a6b976ed794bb8598dbc59993</citedby><cites>FETCH-LOGICAL-c526t-41bf7fc662ef212d043c16f0eafe440720aeb4d2a6b976ed794bb8598dbc59993</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27923,27924</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19737008$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2113524$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Reddy, V.A. (New York State Department of Health, Albany, NY)</creatorcontrib><creatorcontrib>Maley, F</creatorcontrib><title>Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent.
The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the
modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column
chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these
peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing
it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for
Asp-23 in the catalytic process.</description><subject>Acetylglucosaminidase</subject><subject>ACIDE ASPARTIQUE</subject><subject>ACIDO ASPARTICO</subject><subject>actine site amino acid</subject><subject>affinity labelling</subject><subject>Affinity Labels</subject><subject>amino acid residue</subject><subject>Amino Acid Sequence</subject><subject>ASPARTIC ACID</subject><subject>Aspartic Acid - genetics</subject><subject>Base Sequence</subject><subject>beta -fructofuranosidase</subject><subject>BINDING SITE</subject><subject>Binding Sites</subject><subject>Catalysis</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Exact sciences and technology</subject><subject>FRUCTOFURANOSIDASA</subject><subject>FRUCTOFURANOSIDASE</subject><subject>Glycoside Hydrolases - antagonists & inhibitors</subject><subject>Glycoside Hydrolases - genetics</subject><subject>Glycoside Hydrolases - metabolism</subject><subject>Glycosylation</subject><subject>Inositol - analogs & derivatives</subject><subject>Inositol - pharmacology</subject><subject>Kinetics</subject><subject>Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase</subject><subject>Mathematical analysis</subject><subject>Mathematics</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>Peptide Fragments</subject><subject>Potential theory</subject><subject>SACCHAROMYCES CEREVISIAE</subject><subject>Saccharomyces cerevisiae - enzymology</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Sciences and techniques of general use</subject><subject>site-directed mutagenesis</subject><subject>Trypsin</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><recordid>eNpFkV1rFDEUhoModbv6B4RCLlT0YjTJJJnJZSlWCwUvasG7kI-T3chMpk4ylf33Ztyl5uYEzvsBz0HogpJPlFD5ORPCaKOY6D9Q9bHtBe2b7hnaUNK3TSvoz-do8yR5ic5z_kXq44qeoTNGaSsY36B04yGVGKIzJU4JTwGbhI0r8RGaHAvgGXL0C-CY8AFMLvXzCHMxGbA9YBNCTLEc8GAsDDHtqt3j1dj4OIMr4PG4FLODVHPyK_QimCHD69PcovvrLz-uvjW337_eXF3eNk4wWRpObeiCk5JBYJR5wltHZSBgAnBOOkYMWO6ZkVZ1EnynuLW9UL23Tiil2i16f8x9mKffC-Six5gdDINJMC1ZU9EzyVteheIodPOU8wxBP8xxNPNBU6JXzvpuhahXiJoq_Y-z7qrv4lSw2BH8k-sEtu7fnfYmOzOE2SQX8_9w1bUdqZfaordH3T7u9n8qMW3j5PYwaibFWljPSde6N0dZMJM2u7lG3d8pwqVUov0Ldq-bnw</recordid><startdate>19900705</startdate><enddate>19900705</enddate><creator>Reddy, V.A. (New York State Department of Health, Albany, NY)</creator><creator>Maley, F</creator><general>American Society for Biochemistry and Molecular Biology</general><scope>FBQ</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>M7N</scope><scope>M81</scope><scope>P64</scope></search><sort><creationdate>19900705</creationdate><title>Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis</title><author>Reddy, V.A. (New York State Department of Health, Albany, NY) ; Maley, F</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c526t-41bf7fc662ef212d043c16f0eafe440720aeb4d2a6b976ed794bb8598dbc59993</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Acetylglucosaminidase</topic><topic>ACIDE ASPARTIQUE</topic><topic>ACIDO ASPARTICO</topic><topic>actine site amino acid</topic><topic>affinity labelling</topic><topic>Affinity Labels</topic><topic>amino acid residue</topic><topic>Amino Acid Sequence</topic><topic>ASPARTIC ACID</topic><topic>Aspartic Acid - genetics</topic><topic>Base Sequence</topic><topic>beta -fructofuranosidase</topic><topic>BINDING SITE</topic><topic>Binding Sites</topic><topic>Catalysis</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Exact sciences and technology</topic><topic>FRUCTOFURANOSIDASA</topic><topic>FRUCTOFURANOSIDASE</topic><topic>Glycoside Hydrolases - antagonists & inhibitors</topic><topic>Glycoside Hydrolases - genetics</topic><topic>Glycoside Hydrolases - metabolism</topic><topic>Glycosylation</topic><topic>Inositol - analogs & derivatives</topic><topic>Inositol - pharmacology</topic><topic>Kinetics</topic><topic>Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase</topic><topic>Mathematical analysis</topic><topic>Mathematics</topic><topic>Molecular Sequence Data</topic><topic>Mutation</topic><topic>Peptide Fragments</topic><topic>Potential theory</topic><topic>SACCHAROMYCES CEREVISIAE</topic><topic>Saccharomyces cerevisiae - enzymology</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Sciences and techniques of general use</topic><topic>site-directed mutagenesis</topic><topic>Trypsin</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Reddy, V.A. (New York State Department of Health, Albany, NY)</creatorcontrib><creatorcontrib>Maley, F</creatorcontrib><collection>AGRIS</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Reddy, V.A. (New York State Department of Health, Albany, NY)</au><au>Maley, F</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1990-07-05</date><risdate>1990</risdate><volume>265</volume><issue>19</issue><spage>10817</spage><epage>10820</epage><pages>10817-10820</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent.
The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the
modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column
chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these
peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing
it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for
Asp-23 in the catalytic process.</abstract><cop>Bethesda, MD</cop><pub>American Society for Biochemistry and Molecular Biology</pub><pmid>2113524</pmid><doi>10.1016/s0021-9258(19)38518-7</doi><tpages>4</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1990-07, Vol.265 (19), p.10817-10820 |
| issn | 0021-9258 1083-351X |
| language | eng |
| recordid | cdi_proquest_miscellaneous_15826434 |
| source | Elsevier ScienceDirect Journals |
| subjects | Acetylglucosaminidase ACIDE ASPARTIQUE ACIDO ASPARTICO actine site amino acid affinity labelling Affinity Labels amino acid residue Amino Acid Sequence ASPARTIC ACID Aspartic Acid - genetics Base Sequence beta -fructofuranosidase BINDING SITE Binding Sites Catalysis Chromatography, High Pressure Liquid Exact sciences and technology FRUCTOFURANOSIDASA FRUCTOFURANOSIDASE Glycoside Hydrolases - antagonists & inhibitors Glycoside Hydrolases - genetics Glycoside Hydrolases - metabolism Glycosylation Inositol - analogs & derivatives Inositol - pharmacology Kinetics Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase Mathematical analysis Mathematics Molecular Sequence Data Mutation Peptide Fragments Potential theory SACCHAROMYCES CEREVISIAE Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae - genetics Sciences and techniques of general use site-directed mutagenesis Trypsin |
| title | Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis |
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