{"doi":"10.1073/pnas.95.2.466","title":"Vitamin K-dependent carboxylation of the carboxylase","abstract":"<jats:p>\n                    Vitamin K-dependent (VKD) proteins require modification by the VKD-γ-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated\n                    <jats:italic>in vitro</jats:italic>\n                    with\n                    <jats:sup>14</jats:sup>\n                    CO\n                    <jats:sub>2</jats:sub>\n                    and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of\n                    <jats:italic>in vitro</jats:italic>\n                    -modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the\n                    <jats:italic>in vitro</jats:italic>\n                    reaction. Carboxylase carboxylation also was detected in baculovirus(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated\n                    <jats:italic>in vivo</jats:italic>\n                    . Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation\n                    <jats:italic>in vitro</jats:italic>\n                    suggest a possible role for carboxylase carboxylation in factor IX turnover, and\n                    <jats:italic>in vivo</jats:italic>\n                    studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.\n                  </jats:p>","journal":"Proceedings of the National Academy of Sciences","year":1998,"id":684317,"datarank":0.6038027536102726,"base_score":4.02535169073515,"endowment":4.02535169073515,"self_citation_contribution":0.6038027536102726,"citation_network_contribution":0.0,"self_endowment_contribution":0.6038027536102726,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":55,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1787756,"name":"B. Nirmala Pudota","orcid":null,"position":1,"is_corresponding":false},{"id":672037,"name":"Kathleen L. Berkner","orcid":"0000-0001-8054-8081","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Vitamin K-dependent carboxylation of the carboxylase","abstract":"<jats:p>\n                    Vitamin K-dependent (VKD) proteins require modification by the VKD-γ-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated\n                    <jats:italic>in vitro</jats:italic>\n                    with\n                    <jats:sup>14</jats:sup>\n                    CO\n                    <jats:sub>2</jats:sub>\n                    and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of\n                    <jats:italic>in vitro</jats:italic>\n                    -modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the\n                    <jats:italic>in vitro</jats:italic>\n                    reaction. Carboxylase carboxylation also was detected in baculovirus(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated\n                    <jats:italic>in vivo</jats:italic>\n                    . Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation\n                    <jats:italic>in vitro</jats:italic>\n                    suggest a possible role for carboxylase carboxylation in factor IX turnover, and\n                    <jats:italic>in vivo</jats:italic>\n                    studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.\n                  </jats:p>","is_dataset_classified":null,"base_score":4.02535169073515,"endowment":4.02535169073515,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"9435215","pmcid":"PMC18443","openalex_id":"https://openalex.org/W1977265717","authors":[],"funders":[{"funder_name":"NHLBI NIH HHS","grant_id":"R01 HL055666","title":null},{"funder_name":"NHLBI NIH HHS","grant_id":"HL55666","title":null}],"total_grants":2,"fwci":1.4524,"citation_percentile":0.79670155,"influential_citations":0,"citation_trend":[{"year":2012,"count":2},{"year":2013,"count":3},{"year":2014,"count":3},{"year":2015,"count":2},{"year":2018,"count":2},{"year":2019,"count":2},{"year":2021,"count":3},{"year":2022,"count":4},{"year":2023,"count":3},{"year":2024,"count":3},{"year":2025,"count":4},{"year":2026,"count":1}],"oa_status":"closed","license":null,"oa_locations":[{"url":"https://pnas.org/doi/pdf/10.1073/pnas.95.2.466","host_type":"publisher"},{"url":"https://doi.org/10.1073/pnas.95.2.466","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/9435215","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/18443","host_type":"repository"}],"fields_of_study":["Vitamin K Research Studies","Vitamin C and Antioxidants Research","Phagocytosis and Immune Regulation","Carbon-Carbon Ligases","Cell Line","Chromatography, High Pressure Liquid","Humans","Kinetics","Substrate Specificity","Vitamin K"],"mesh_terms":["Cell Line","Chromatography, High Pressure Liquid","Humans","Kinetics","Substrate Specificity","Vitamin K","Carbon-Carbon Ligases"],"keywords":["Carboxylation","Pyruvate carboxylase","Biochemistry","Vitamin","Chemistry","Enzyme","Biology"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Clean water and sanitation"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-18T13:46:51.889275Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}