Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid

Shen, Sida; Butrin, Arseniy; Beaupre, Brett A.; Ferreira, Glaucio M.; Doubleday, Peter F.; Grass, Daniel H.; Zhu, Wei; Kelleher, Neil L.; Moran, Graham R.; Liu, Dali; Silverman, Richard B.

doi:10.3390/molecules28031133

Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid

Journal Article · Sun Jan 22 23:00:00 EST 2023 · Molecules

DOI:https://doi.org/10.3390/molecules28031133· OSTI ID:2423630

^[1]; Butrin, Arseniy ^[2]; Beaupre, Brett A. ^[2]; ^[3]; ^[1]; ^[1]; Zhu, Wei ^[1]; Kelleher, Neil L. ^[1]; Moran, Graham R. ^[2]; ^[2]; ^[1]

Northwestern Univ., Evanston, IL (United States)
Loyola University Chicago, IL (United States)
Univ. of Sao Paulo (Brazil)

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (k_inact/K_I) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pK_a and electrostatic potential calculations were performed to further support the notion that the α,β-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,β-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 2423630

Journal Information:: Molecules, Journal Name: Molecules Journal Issue: 3 Vol. 28; ISSN 1420-3049

Publisher:: MDPICopyright Statement

Country of Publication:: United States

Language:: English

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