{"corpus_id":25883292,"paper_sha":"d0ff9034a36f22c3db8f3508f1f4b5bcbed8b0b5","doi":"10.1016/s0021-9258(17)41891-6","arxiv_id":null,"pmid":7906268,"pmcid":null,"mag_id":2143729650,"dblp_id":null,"acl_id":null,"title":"Functional roles of Trp337 and Glu632 in Clostridium glucoamylase, as determined by chemical modification, mutagenesis, and the stopped-flow method.","year":1994,"publication_date":"1994-02-04","venue":"Journal of Biological Chemistry","journal":{"name":"The Journal of biological chemistry","pages":"\n          3503-10\n        ","volume":"269 5"},"journal_issn":null,"journal_title":null,"publication_types":["JournalArticle","Study"],"pubmed_pub_types":["Comparative Study","Journal Article","Research Support, Non-U.S. Gov't"],"s2_fields_of_study":["Biology","Medicine","Chemistry"],"reference_count":0,"citation_count":17,"influential_citation_count":2,"is_open_access":false,"arxiv_categories":null,"arxiv_license":null,"arxiv_journal_ref":null,"mesh_headings":[{"d":"Acarbose","mj":false,"ui":"D020909"},{"d":"Amino Acid Sequence","mj":false,"ui":"D000595"},{"d":"Base Sequence","mj":false,"ui":"D001483"},{"d":"Bromosuccinimide","mj":false,"qs":[{"q":"pharmacology","mj":true,"ui":"Q000494"}],"ui":"D001974"},{"d":"Clostridium","mj":false,"qs":[{"q":"enzymology","mj":true,"ui":"Q000201"}],"ui":"D003013"},{"d":"Glucan 1,4-alpha-Glucosidase","mj":false,"qs":[{"q":"chemistry","mj":true,"ui":"Q000737"},{"q":"genetics","mj":false,"ui":"Q000235"},{"q":"metabolism","mj":true,"ui":"Q000378"}],"ui":"D005087"},{"d":"Glutamates","mj":true,"ui":"D005971"},{"d":"Glutamic Acid","mj":false,"ui":"D018698"},{"d":"Hypoglycemic Agents","mj":false,"qs":[{"q":"pharmacology","mj":false,"ui":"Q000494"}],"ui":"D007004"},{"d":"Kinetics","mj":false,"ui":"D007700"},{"d":"Mathematics","mj":false,"ui":"D008433"},{"d":"Models, Theoretical","mj":false,"ui":"D008962"},{"d":"Molecular Sequence Data","mj":false,"ui":"D008969"},{"d":"Mutagenesis, Site-Directed","mj":false,"ui":"D016297"},{"d":"Oligodeoxyribonucleotides","mj":false,"ui":"D009838"},{"d":"Point Mutation","mj":true,"ui":"D017354"},{"d":"Protein Conformation","mj":false,"ui":"D011487"},{"d":"Spectrometry, Fluorescence","mj":false,"ui":"D013050"},{"d":"Substrate Specificity","mj":false,"ui":"D013379"},{"d":"Trisaccharides","mj":false,"qs":[{"q":"pharmacology","mj":false,"ui":"Q000494"}],"ui":"D014312"},{"d":"Tryptophan","mj":true,"ui":"D014364"}],"chemicals":[{"n":"Glutamates","ui":"D005971","reg":"0"},{"n":"Hypoglycemic Agents","ui":"D007004","reg":"0"},{"n":"Oligodeoxyribonucleotides","ui":"D009838","reg":"0"},{"n":"Trisaccharides","ui":"D014312","reg":"0"},{"n":"Glutamic Acid","ui":"D018698","reg":"3KX376GY7L"},{"n":"Tryptophan","ui":"D014364","reg":"8DUH1N11BX"},{"n":"Glucan 1,4-alpha-Glucosidase","ui":"D005087","reg":"EC 3.2.1.3"},{"n":"Bromosuccinimide","ui":"D001974","reg":"K8G1F2UCJF"},{"n":"Acarbose","ui":"D020909","reg":"T58MSI464G"}],"comments_corrections":null,"source_flags":5,"s2_open_access_pdf_url":null,"s2_open_access_landing_url":null,"s2_open_access_license":null,"s2_open_access_status":null,"pmc_open_access_pdf_url":null,"pmc_open_access_landing_url":null,"pmc_open_access_license":null,"pmc_open_access_status":null,"unpaywall_open_access_pdf_url":null,"unpaywall_open_access_landing_url":null,"unpaywall_open_access_license":null,"unpaywall_open_access_status":null,"abstract":"Chemical modification of glucoamylase (EC 3.2.1.3) from Clostridium sp. G0005 (CGA) with N-bromosuccinimide (NBS) was carried out in the presence or absence of an inhibitor, acarbose. CGA lost its catalytic activity through NBS oxidation in the absence of acarbose. The absorbance change at 280 nm suggested that acarbose protects about 2 Trp residues from NBS oxidation. We performed peptide mapping analysis to identify the protected Trp residues, and Trp321, Trp337, Trp433, and Trp569 were identified as candidates to be protected by acarbose. These 4 Trp residues were replaced by site-directed mutagenesis with Phe. The Trp337-->Phe mutant showed very weak catalytic activity, so Trp337 is proposed as an important residue for the catalytic activity. Further, we constructed a Glu632-->Gln mutant. Glu632 is the putative catalytic base. The presteady-state kinetics of the Trp337-->Phe and Glu632-->Gln mutants and the wild-type CGA were investigated using maltotriose as a substrate. The reaction of wild-type CGA can be explained as one involving three intermediates. On the other hand, the two mutants' reactions are explained by a two-step mechanism lacking the third intermediate. Trp337 and Glu632 appear to be crucial for the formation of the third intermediate in the wild-type reaction, which precedes the transition state.","claims":[{"public_id":"cl_e44ba69c394e6795323aab8daf1118aa","status":"active","text":"Chemical modification with N-bromosuccinimide in the absence of acarbose caused Clostridium glucoamylase to lose catalytic activity, and acarbose protected about 2 Trp residues from NBS oxidation.","confidence":0.9,"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_e44ba69c394e6795323aab8daf1118aa"},{"public_id":"cl_9b4d6c4d3268976cb4ce91e66cc2c576","status":"active","text":"Peptide mapping identified Trp321, Trp337, Trp433, and Trp569 as candidate Trp residues protected by acarbose; the Trp337→Phe mutant showed very weak catalytic activity, indicating Trp337 is important for catalytic activity.","confidence":0.9,"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_9b4d6c4d3268976cb4ce91e66cc2c576"},{"public_id":"cl_1b2e5e414c4aaab23be16d8961705d2a","status":"active","text":"The Glu632→Gln mutant was constructed because Glu632 is the putative catalytic base; presteady-state kinetics with maltotriose showed that wild-type CGA follows a three-intermediate mechanism, while the Trp337→Phe and Glu632→Gln mutants follow a two-step mechanism lacking the third intermediate.","confidence":0.95,"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_1b2e5e414c4aaab23be16d8961705d2a"},{"public_id":"cl_86f3a20d72ab57a3be5e7ef0c678d318","status":"active","text":"Trp337 and Glu632 appear crucial for formation of the third intermediate in the wild-type reaction, which precedes the transition state.","confidence":0.95,"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/claims/cl_86f3a20d72ab57a3be5e7ef0c678d318"}],"concepts":[{"public_id":"co_1df66449c5e3ba61290e3e01fbe8b9d6","status":"active","name":"Trp569","description":"Tryptophan residue at position 569 in CGA, identified as a candidate protected by acarbose.","types":["residue"],"aliases":[],"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_1df66449c5e3ba61290e3e01fbe8b9d6"},{"public_id":"co_20ce8755f390b034daee3449f33b24a9","status":"active","name":"third intermediate","description":"The third intermediate in the wild-type CGA reaction that precedes the transition state and whose formation depends on Trp337 and Glu632.","types":["intermediate"],"aliases":[],"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_20ce8755f390b034daee3449f33b24a9"},{"public_id":"co_24abb011bc978c0a509ebcf54267e244","status":"active","name":"acarbose protection","description":"Protection of Trp residues from NBS oxidation by the inhibitor acarbose, used to identify functionally important Trp residues.","types":["inhibitor","method"],"aliases":[],"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_24abb011bc978c0a509ebcf54267e244"},{"public_id":"co_42aac2ffc9145e6e60fe49b2f8c9f8e2","status":"active","name":"three-intermediate mechanism","description":"Reaction mechanism of wild-type CGA involving three intermediates as deduced from presteady-state kinetics.","types":["mechanism"],"aliases":[],"contributors":[{"id":32,"public_id":"7c402c1b98","public_label":"뀨 (7c402c1b98)","roles":["extraction"],"url":"https://sah.borca.ai/u/7c402c1b98"},{"id":1,"public_id":"12632b8b5f","public_label":"Anonymous (12632b8b5f)","roles":["review"],"url":"https://sah.borca.ai/u/12632b8b5f"}],"url":"https://sah.borca.ai/concepts/co_42aac2ffc9145e6e60fe49b2f8c9f8e2"},{"public_id":"co_4df3c144917575cccb202afe1369c3a3","status":"active","name":"Clostridium glucoamylase","description":"Glucoamylase (EC 3.2.1.3) from Clostridium sp. 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