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dc.contributor.author
Shen, Sida
dc.contributor.author
Butrin, Arseniy
dc.contributor.author
Doubleday, Peter F.
dc.contributor.author
Melani, Rafael D.
dc.contributor.author
Beaupre, Brett A.
dc.contributor.author
Tavares, Mauricio T.
dc.contributor.author
Ferreira, Glaucio M.
dc.contributor.author
Kelleher, Neil L.
dc.contributor.author
Moran, Graham R.
dc.contributor.author
Liu, Dali
dc.contributor.author
Silverman, Richard B.
dc.date.accessioned
2021-07-20T21:16:33Z
dc.date.available
2021-07-15T02:29:27Z
dc.date.available
2021-07-20T21:16:33Z
dc.date.issued
2021-06-16
dc.identifier.issn
0002-7863
dc.identifier.issn
1520-5126
dc.identifier.other
10.1021/jacs.1c02456
en_US
dc.identifier.uri
http://hdl.handle.net/20.500.11850/494645
dc.description.abstract
The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.
en_US
dc.language.iso
en
en_US
dc.publisher
American Chemical Society
en_US
dc.title
Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase
en_US
dc.type
Journal Article
dc.date.published
2021-06-07
ethz.journal.title
Journal of the American Chemical Society
ethz.journal.volume
143
en_US
ethz.journal.issue
23
en_US
ethz.journal.abbreviated
J. Am. Chem. Soc.
ethz.pages.start
8689
en_US
ethz.pages.end
8703
en_US
ethz.identifier.wos
ethz.publication.place
Washington, DC
en_US
ethz.publication.status
published
en_US
ethz.date.deposited
2021-07-15T02:30:04Z
ethz.source
WOS
ethz.eth
yes
en_US
ethz.availability
Metadata only
en_US
ethz.rosetta.installDate
2021-07-20T21:16:39Z
ethz.rosetta.lastUpdated
2022-03-29T10:32:58Z
ethz.rosetta.versionExported
true
ethz.COinS
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