{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,5,22]],"date-time":"2025-05-22T17:46:40Z","timestamp":1747936000341},"reference-count":0,"publisher":"Portland Press Ltd.","issue":"3","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[1997,2,1]]},"abstract":"<jats:p>In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase\/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000-and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase.<\/jats:p>","DOI":"10.1042\/bj3210623","type":"journal-article","created":{"date-parts":[[2015,8,10]],"date-time":"2015-08-10T21:52:36Z","timestamp":1439243556000},"page":"623-627","update-policy":"http:\/\/dx.doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase\/fructose-2,6-bisphosphatase"],"prefix":"10.1042","volume":"321","author":[{"given":"Luc","family":"BERTRAND","sequence":"first","affiliation":[{"name":"Hormone and Metabolic Research Unit, University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 75, B-1200 Brussels, Belgium"},{"name":"Facult\u00e9s Universitaires Notre-Dame de la Paix, Department of Biology, Rue de Bruxelles 61, B-5000 Namur, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Johan","family":"DEPREZ","sequence":"additional","affiliation":[{"name":"Hormone and Metabolic Research Unit, University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 75, B-1200 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Didier","family":"VERTOMMEN","sequence":"additional","affiliation":[{"name":"Hormone and Metabolic Research Unit, University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 75, B-1200 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Attilio DI","family":"PIETRO","sequence":"additional","affiliation":[{"name":"Institut de Biologie et Chimie des Prot\u00e9ines, Centre National de la Recherche Scientifique, Passage du Vercors, 7, F-69367 Lyon cedex 07, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Louis","family":"HUE","sequence":"additional","affiliation":[{"name":"Hormone and Metabolic Research Unit, University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 75, B-1200 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark H.","family":"RIDER","sequence":"additional","affiliation":[{"name":"Hormone and Metabolic Research Unit, University of Louvain Medical School and International Institute of Cellular and Molecular Pathology, Avenue Hippocrate, 75, B-1200 Brussels, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/321\/3\/623\/623629\/bj3210623.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/321\/3\/623\/623629\/bj3210623.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,24]],"date-time":"2021-11-24T00:36:18Z","timestamp":1637714178000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/321\/3\/623\/34149\/Site-directed-mutagenesis-of-Lys-174-Asp-179-and"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1997,2,1]]},"references-count":0,"journal-issue":{"issue":"3","published-print":{"date-parts":[[1997,2,1]]}},"URL":"https:\/\/doi.org\/10.1042\/bj3210623","relation":{},"ISSN":["0264-6021","1470-8728"],"issn-type":[{"value":"0264-6021","type":"print"},{"value":"1470-8728","type":"electronic"}],"subject":[],"published":{"date-parts":[[1997,2,1]]}}}