The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense
The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-depen...
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| Veröffentlicht in: | European journal of biochemistry 1991-11, Vol.202 (1), p.181-189 |
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| container_title | European journal of biochemistry |
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| creator | Vanoni, M.A. (Universita degli Studi di Milano, Milano, Italy) Nuzzi, L Rescigno, M Zanetti, G Curti, B |
| description | The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. and Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity |
| doi_str_mv | 10.1111/j.1432-1033.1991.tb16361.x |
| format | Article |
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The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. and Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity</description><identifier>ISSN: 0014-2956</identifier><identifier>EISSN: 1432-1033</identifier><identifier>DOI: 10.1111/j.1432-1033.1991.tb16361.x</identifier><identifier>PMID: 1935975</identifier><identifier>CODEN: EJBCAI</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Publishing Ltd</publisher><subject>ACTIVIDAD CATALITICA ; ACTIVITE CATALYTIQUE ; Ammonia - pharmacology ; AMMONIAC ; AMONIACO ; AZOSPIRILLUM ; Azospirillum brasilense ; Azospirillum brasilense - enzymology ; Bacteriology ; Biological and medical sciences ; Fundamental and applied biological sciences. Psychology ; Glutamate Synthase - antagonists & inhibitors ; Glutamate Synthase - metabolism ; GLUTAMINA ; GLUTAMINE ; Glutamine - metabolism ; Glutamine - pharmacology ; INHIBICION ; INHIBIDORES DE ENZIMAS ; INHIBITEUR D'ENZYME ; INHIBITION ; Ketoglutaric Acids - metabolism ; Kinetics ; MEDIO DE CULTIVO ; Metabolism. Enzymes ; Microbiology ; MILIEU DE CULTURE ; NADP - metabolism ; Oxidation-Reduction ; OXIDORREDUCTASAS ; OXYDOREDUCTASE ; Spectrophotometry ; Substrate Specificity ; Tritium</subject><ispartof>European journal of biochemistry, 1991-11, Vol.202 (1), p.181-189</ispartof><rights>1992 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5011-6715592fb41118bc2c84f81bd38264e4d95e380860a99c3088b44e3d654b26a83</citedby><cites>FETCH-LOGICAL-c5011-6715592fb41118bc2c84f81bd38264e4d95e380860a99c3088b44e3d654b26a83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=5196644$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/1935975$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vanoni, M.A. (Universita degli Studi di Milano, Milano, Italy)</creatorcontrib><creatorcontrib>Nuzzi, L</creatorcontrib><creatorcontrib>Rescigno, M</creatorcontrib><creatorcontrib>Zanetti, G</creatorcontrib><creatorcontrib>Curti, B</creatorcontrib><title>The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense</title><title>European journal of biochemistry</title><addtitle>Eur J Biochem</addtitle><description>The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. and Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity</description><subject>ACTIVIDAD CATALITICA</subject><subject>ACTIVITE CATALYTIQUE</subject><subject>Ammonia - pharmacology</subject><subject>AMMONIAC</subject><subject>AMONIACO</subject><subject>AZOSPIRILLUM</subject><subject>Azospirillum brasilense</subject><subject>Azospirillum brasilense - enzymology</subject><subject>Bacteriology</subject><subject>Biological and medical sciences</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glutamate Synthase - antagonists & inhibitors</subject><subject>Glutamate Synthase - metabolism</subject><subject>GLUTAMINA</subject><subject>GLUTAMINE</subject><subject>Glutamine - metabolism</subject><subject>Glutamine - pharmacology</subject><subject>INHIBICION</subject><subject>INHIBIDORES DE ENZIMAS</subject><subject>INHIBITEUR D'ENZYME</subject><subject>INHIBITION</subject><subject>Ketoglutaric Acids - metabolism</subject><subject>Kinetics</subject><subject>MEDIO DE CULTIVO</subject><subject>Metabolism. Enzymes</subject><subject>Microbiology</subject><subject>MILIEU DE CULTURE</subject><subject>NADP - metabolism</subject><subject>Oxidation-Reduction</subject><subject>OXIDORREDUCTASAS</subject><subject>OXYDOREDUCTASE</subject><subject>Spectrophotometry</subject><subject>Substrate Specificity</subject><subject>Tritium</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1991</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqVkUtv1DAUhS0EKtPCH0BCshDqLsE3dhybDSpVC0iVWLRdW7bjzHjIY7Ad0fTXk5BRWSK88eJ893UOQu-A5DC_D_scGC0yIJTmICXkyQCnHPKHZ2jzJD1HG0KAZYUs-Ut0GuOeEMIlr07QCUhayqrcoO3dzuEfvnfJW9w5u9O9jx0eGpxmIThtkx_6iK1Oup0eXY3N9EfatmPSnU4Ox6lPOx0dbsLQ4YvHIR588G07dtgEHX3r-uheoReNbqN7ffzP0P311d3l1-zm-5dvlxc3mS0JQMYrKEtZNIbNdwpjCytYI8DUVBScOVbL0lFBBCdaSkuJEIYxR2teMlNwLegZOl_7HsLwc3Qxqc5H69pW924Yo6oKxkQF8E8QOJmxaun4cQVtGGIMrlGH4DsdJgVELXGovVo8V4vnaolDHeNQD3Px2-OU0XSu_lu6-j_r74-6jla3TdC99fEJK0FyztiMfVqxX7Ob038soK6vPt-CWM59s3Zo9KD0NsxD7m8lVBUrOP0Nrtivgg</recordid><startdate>19911115</startdate><enddate>19911115</enddate><creator>Vanoni, M.A. (Universita degli Studi di Milano, Milano, Italy)</creator><creator>Nuzzi, L</creator><creator>Rescigno, M</creator><creator>Zanetti, G</creator><creator>Curti, B</creator><general>Blackwell Publishing Ltd</general><general>Blackwell</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>M81</scope><scope>P64</scope><scope>7X8</scope></search><sort><creationdate>19911115</creationdate><title>The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense</title><author>Vanoni, M.A. (Universita degli Studi di Milano, Milano, Italy) ; Nuzzi, L ; Rescigno, M ; Zanetti, G ; Curti, B</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5011-6715592fb41118bc2c84f81bd38264e4d95e380860a99c3088b44e3d654b26a83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1991</creationdate><topic>ACTIVIDAD CATALITICA</topic><topic>ACTIVITE CATALYTIQUE</topic><topic>Ammonia - pharmacology</topic><topic>AMMONIAC</topic><topic>AMONIACO</topic><topic>AZOSPIRILLUM</topic><topic>Azospirillum brasilense</topic><topic>Azospirillum brasilense - enzymology</topic><topic>Bacteriology</topic><topic>Biological and medical sciences</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glutamate Synthase - antagonists & inhibitors</topic><topic>Glutamate Synthase - metabolism</topic><topic>GLUTAMINA</topic><topic>GLUTAMINE</topic><topic>Glutamine - metabolism</topic><topic>Glutamine - pharmacology</topic><topic>INHIBICION</topic><topic>INHIBIDORES DE ENZIMAS</topic><topic>INHIBITEUR D'ENZYME</topic><topic>INHIBITION</topic><topic>Ketoglutaric Acids - metabolism</topic><topic>Kinetics</topic><topic>MEDIO DE CULTIVO</topic><topic>Metabolism. Enzymes</topic><topic>Microbiology</topic><topic>MILIEU DE CULTURE</topic><topic>NADP - metabolism</topic><topic>Oxidation-Reduction</topic><topic>OXIDORREDUCTASAS</topic><topic>OXYDOREDUCTASE</topic><topic>Spectrophotometry</topic><topic>Substrate Specificity</topic><topic>Tritium</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Vanoni, M.A. (Universita degli Studi di Milano, Milano, Italy)</creatorcontrib><creatorcontrib>Nuzzi, L</creatorcontrib><creatorcontrib>Rescigno, M</creatorcontrib><creatorcontrib>Zanetti, G</creatorcontrib><creatorcontrib>Curti, B</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>Biochemistry Abstracts 3</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vanoni, M.A. (Universita degli Studi di Milano, Milano, Italy)</au><au>Nuzzi, L</au><au>Rescigno, M</au><au>Zanetti, G</au><au>Curti, B</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense</atitle><jtitle>European journal of biochemistry</jtitle><addtitle>Eur J Biochem</addtitle><date>1991-11-15</date><risdate>1991</risdate><volume>202</volume><issue>1</issue><spage>181</spage><epage>189</epage><pages>181-189</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><coden>EJBCAI</coden><abstract>The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. and Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>1935975</pmid><doi>10.1111/j.1432-1033.1991.tb16361.x</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | European journal of biochemistry, 1991-11, Vol.202 (1), p.181-189 |
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| subjects | ACTIVIDAD CATALITICA ACTIVITE CATALYTIQUE Ammonia - pharmacology AMMONIAC AMONIACO AZOSPIRILLUM Azospirillum brasilense Azospirillum brasilense - enzymology Bacteriology Biological and medical sciences Fundamental and applied biological sciences. Psychology Glutamate Synthase - antagonists & inhibitors Glutamate Synthase - metabolism GLUTAMINA GLUTAMINE Glutamine - metabolism Glutamine - pharmacology INHIBICION INHIBIDORES DE ENZIMAS INHIBITEUR D'ENZYME INHIBITION Ketoglutaric Acids - metabolism Kinetics MEDIO DE CULTIVO Metabolism. Enzymes Microbiology MILIEU DE CULTURE NADP - metabolism Oxidation-Reduction OXIDORREDUCTASAS OXYDOREDUCTASE Spectrophotometry Substrate Specificity Tritium |
| title | The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense |
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