{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,2,21]],"date-time":"2025-02-21T11:53:49Z","timestamp":1740138829198,"version":"3.37.3"},"reference-count":30,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2007,11,14]],"date-time":"2007-11-14T00:00:00Z","timestamp":1194998400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["GM26237"],"award-info":[{"award-number":["GM26237"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Institutes of Health, National Center for Research Resources","award":["P41RR01209"],"award-info":[{"award-number":["P41RR01209"]}]},{"name":"Department of Energy, Office of Biological and Environmental Research"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Proteins"],"published-print":{"date-parts":[[2008,5,15]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The mechanism of domain closure and the allosteric transition of <jats:italic>Escherichia coli<\/jats:italic> aspartate transcarbamoylase (ATCase) are investigated using <jats:sc>L<\/jats:sc>\u2010Asn, in the presence of carbamoyl phosphate (CP), and N\u2010phosphonacetyl\u2010<jats:sc>L<\/jats:sc>\u2010asparagine (PASN). ATCase was found to catalyze the carbamoylation of <jats:sc>L<\/jats:sc>\u2010Asn with a K<jats:sub>m<\/jats:sub> of 122 mM and a maximal velocity 10\u2010fold lower than observed with the natural substrate, <jats:sc>L<\/jats:sc>\u2010Asp. As opposed to <jats:sc>L<\/jats:sc>\u2010Asp, no cooperativity was observed with respect to <jats:sc>L<\/jats:sc>\u2010Asn. Time\u2010resolved small\u2010angle X\u2010ray scattering (SAXS) and fluorescence experiments revealed that the combination of CP and <jats:sc>L<\/jats:sc>\u2010Asn did not convert the enzyme from the T to the R state. PASN was found to be a potent inhibitor of ATCase exhibiting a K<jats:sub>D<\/jats:sub> of 8.8 \u03bcM. SAXS experiments showed that PASN was able to convert the entire population of molecules to the R state. Analysis of the crystal structure of the enzyme in the presence of PASN revealed that the binding of PASN was similar to that of the R\u2010state complex of ATCase with N\u2010phosphonaceyl\u2010<jats:sc>L<\/jats:sc>\u2010aspartate, another potent inhibitor of the enzyme. The linking of CP and <jats:sc>L<\/jats:sc>\u2010Asn into one molecule, PASN, correctly orients the asparagine moiety in the active site to induce domain closure and the allosteric transition. This entropic effect allows for the high affinity binding of PASN. However, the binding of <jats:sc>L<\/jats:sc>\u2010Asn, in the presence of a saturating concentration of CP, does not induce the closure of the two domains of the catalytic chain, nor does the enzyme undergo the transition to the high\u2010activity high\u2010 affinity R structure. These results imply that Arg229, which interacts with the \u03b2\u2010carboxylate of <jats:sc>L<\/jats:sc>\u2010Asp, plays a critical role in the orientation of <jats:sc>L<\/jats:sc>\u2010Asp in the active site and demonstrates the requirement of the \u03b2\u2010carboxylate of <jats:sc>L<\/jats:sc>\u2010Asp in the mechanism of domain closure and the allosteric transition in <jats:italic>E. coli<\/jats:italic> ATCase. Proteins 2008. \u00a9 2007 Wiley\u2010Liss, Inc.<\/jats:p>","DOI":"10.1002\/prot.21760","type":"journal-article","created":{"date-parts":[[2007,11,14]],"date-time":"2007-11-14T18:20:33Z","timestamp":1195064433000},"page":"1088-1096","source":"Crossref","is-referenced-by-count":3,"title":["Use of <scp>L<\/scp>\u2010asparagine and <i>N<\/i>\u2010phosphonacetyl\u2010<scp>L<\/scp>\u2010asparagine to investigate the linkage of catalysis and homotropic cooperativity in <i>E. coli<\/i> aspartate transcarbomoylase"],"prefix":"10.1002","volume":"71","author":[{"given":"James P.","family":"Cardia","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joby","family":"Eldo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiarong","family":"Xia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth M.","family":"O'Day","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hiro","family":"Tsuruta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kimberly R.","family":"Gryncel","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Evan R.","family":"Kantrowitz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2007,11,14]]},"reference":[{"key":"e_1_2_6_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-9861(87)90498-X"},{"key":"e_1_2_6_3_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0503742102"},{"key":"e_1_2_6_4_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.82.6.1643"},{"key":"e_1_2_6_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(87)90265-8"},{"key":"e_1_2_6_6_2","doi-asserted-by":"crossref","first-page":"891","DOI":"10.1016\/S0021-9258(18)60389-8","article-title":"Enzymology of control by feedback inhibition","volume":"237","author":"Gerhart JC","year":"1962","journal-title":"J Biol Chem"},{"key":"e_1_2_6_7_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.86.1.46"},{"key":"e_1_2_6_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(88)90365-8"},{"key":"e_1_2_6_9_2","first-page":"1175","article-title":"Aspartate transcarbamylase. Stereospecific restrictions on the binding site for L\u2010aspartate","volume":"245","author":"Davies G","year":"1970","journal-title":"The J Biol Chem"},{"key":"e_1_2_6_10_2","doi-asserted-by":"publisher","DOI":"10.1021\/jm0607294"},{"key":"e_1_2_6_11_2","doi-asserted-by":"crossref","first-page":"14712","DOI":"10.1016\/S0021-9258(17)38630-1","article-title":"Superproduction and rapid purification of E. coli aspartate transcarbamoylase and its catalytic subunit under extreme derepression of the pyrimidine pathway","volume":"260","author":"Nowlan SF","year":"1985","journal-title":"J Biol Chem"},{"key":"e_1_2_6_12_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M304018200"},{"key":"e_1_2_6_13_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(76)90527-3"},{"key":"e_1_2_6_14_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1462-5822.2007.00901.x"},{"key":"e_1_2_6_15_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1749-6632.1964.tb14213.x"},{"key":"e_1_2_6_16_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1749-6632.1964.tb14207.x"},{"key":"e_1_2_6_17_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2005.03.073"},{"key":"e_1_2_6_18_2","first-page":"1718","article-title":"The finer things in X\u2010ray diffraction data collection","volume":"55","author":"Pflugrath JW","year":"1999","journal-title":"Acta Cryst"},{"key":"e_1_2_6_19_2","doi-asserted-by":"publisher","DOI":"10.1002\/(SICI)1097-0134(19991201)37:4<729::AID-PROT21>3.0.CO;2-F"},{"key":"e_1_2_6_20_2","first-page":"905","article-title":"Crystallography and NMR system (CNS): a new software system for macromolecular structure determination","volume":"54","author":"Br\u00fcnger AT","year":"1998","journal-title":"Acta Cryst"},{"key":"e_1_2_6_21_2","doi-asserted-by":"publisher","DOI":"10.1107\/S0907444904019158"},{"key":"e_1_2_6_22_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(81)90594-7"},{"key":"e_1_2_6_23_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00458a015"},{"key":"e_1_2_6_24_2","doi-asserted-by":"publisher","DOI":"10.1016\/0022-2836(85)90190-1"},{"key":"e_1_2_6_25_2","doi-asserted-by":"publisher","DOI":"10.1016\/0014-5793(90)80706-O"},{"key":"e_1_2_6_26_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00346a025"},{"key":"e_1_2_6_27_2","doi-asserted-by":"crossref","first-page":"9624","DOI":"10.1016\/S0021-9258(17)39283-9","article-title":"Substrate specificity of aspartate transcarbamylase. Interaction of the enzyme with analogs of aspartate and succinate","volume":"260","author":"Foote J","year":"1985","journal-title":"J Bio Chem"},{"key":"e_1_2_6_28_2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00430a029"},{"volume-title":"Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding","year":"1998","author":"Fersht AR","key":"e_1_2_6_29_2"},{"key":"e_1_2_6_30_2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.5560040212"},{"key":"e_1_2_6_31_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmb.2004.06.002"}],"container-title":["Proteins: Structure, Function, and Bioinformatics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fprot.21760","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fprot.21760","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/prot.21760","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,10,15]],"date-time":"2023-10-15T12:27:31Z","timestamp":1697372851000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1002\/prot.21760"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2007,11,14]]},"references-count":30,"journal-issue":{"issue":"3","published-print":{"date-parts":[[2008,5,15]]}},"alternative-id":["10.1002\/prot.21760"],"URL":"https:\/\/doi.org\/10.1002\/prot.21760","archive":["Portico"],"relation":{},"ISSN":["0887-3585","1097-0134"],"issn-type":[{"type":"print","value":"0887-3585"},{"type":"electronic","value":"1097-0134"}],"subject":[],"published":{"date-parts":[[2007,11,14]]}}}