{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T00:56:47Z","timestamp":1768525007380,"version":"3.49.0"},"posted":{"date-parts":[[2025,9,17]]},"group-title":"Biochemistry","reference-count":51,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,17]],"date-time":"2025-09-17T00:00:00Z","timestamp":1758067200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"NIH","award":["R35 GM152206"],"award-info":[{"award-number":["R35 GM152206"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,9,17]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  The glutaminase (GLS) isoforms KGA and GAC are expressed in neurons where they hydrolyze glutamine to produce the excitatory neurotransmitter glutamate. Two\n                  <jats:italic>de novo<\/jats:italic>\n                  gain-of-function mutants of GLS, S482C and H461L, were recently identified in patients with developmental delay, epilepsy, and infantile cataract. These patients exhibited high glutamate and low glutamine concentrations in the brain, suggesting that the GLS mutants have abnormal enzymology. Here, we examined the enzymatic properties of these GLS mutants and found that they exhibit a total (S482C) or partial (H461L) loss of glutamate product inhibition, lifting this restriction on glutamate accumulation. The mutant enzymes also no longer require the anionic activator phosphate to stimulate enzymatic activity or induce filament formation. Structural analysis of the S482C GAC mutant shows the mutation shifts the key catalytic residue Y466 into the catalytically competent position and disrupts a key hydrogen bond between it and the glutamate product, explaining how the S482C mutant has enzymatic activity in the absence of phosphate and is insensitive to glutamate product inhibition. These results shed new light on the mechanism of phosphate activation and glutamate product inhibition of GLS and show that loss of these enzymatic properties disrupts glutamate homeostasis in the brain and causes neurological disease.\n                <\/jats:p>","DOI":"10.1101\/2025.09.14.674662","type":"posted-content","created":{"date-parts":[[2025,9,17]],"date-time":"2025-09-17T23:55:13Z","timestamp":1758153313000},"source":"Crossref","is-referenced-by-count":0,"title":["Structure and enzymology of glutaminase mutants that disrupt glutamine-glutamate homeostasis and cause neurological disease"],"prefix":"10.64898","author":[{"given":"Cl\u00e9a S.","family":"Crane","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Thora K.","family":"McIssac","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3624-0113","authenticated-orcid":false,"given":"Shawn K.","family":"Milano","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4512-5897","authenticated-orcid":false,"given":"Richard A.","family":"Cerione","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7117-3684","authenticated-orcid":false,"given":"Scott M.","family":"Ulrich","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025092018300860000_2025.09.14.674662v1.1","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.nu.15.070195.001025"},{"key":"2025092018300860000_2025.09.14.674662v1.2","doi-asserted-by":"publisher","DOI":"10.4155\/fmc-2016-0190"},{"key":"2025092018300860000_2025.09.14.674662v1.3","doi-asserted-by":"publisher","DOI":"10.1111\/j.1471-4159.2006.03913.x"},{"key":"2025092018300860000_2025.09.14.674662v1.4","doi-asserted-by":"crossref","unstructured":"Parpura, V. Glutamate and ATP at the Interface of Metabolism and Signaling in the Brain. (Springer International Publishing AG, Cham, 2014).","DOI":"10.1007\/978-3-319-08894-5"},{"key":"2025092018300860000_2025.09.14.674662v1.5","doi-asserted-by":"publisher","DOI":"10.1111\/jnc.15811"},{"key":"2025092018300860000_2025.09.14.674662v1.6","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1002\/acn3.522","article-title":"GLS loss of function causes autosomal recessive spastic ataxia and optic atrophy","volume":"5","year":"2018","journal-title":"Ann. Clin. Transl. 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