Remarkable and Unexpected Mechanism for (S)-3-Amino-4-(difluoromethylenyl)cyclohex-1-ene-1-carboxylic Acid as a Selective Inactivator of Human Ornithine Aminotransferase

Wei Zhu, Peter F. Doubleday, Arseniy Butrin, Pathum M. Weerawarna, Rafael D. Melani, Daniel S. Catlin, Timothy A. Dwight, Dali Liu, Neil L. Kelleher, Richard B. Silverman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Human ornithine aminotransferase (hOAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of hOAT by compound 10 exhibited potent in vivo antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1S,3S)-3-amino-4-(difluoromethylene)cyclopentane-1-carboxylic acid (5), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, 14 was identified as a new selective hOAT inactivator, which demonstrated a potency 22× greater than that of 10. Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of hOAT-14 and the formation of a remarkable final adduct (32′) in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate 27′ and elucidate the order of the two covalent bonds that formed, leading to 32′. The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of 14 by hOAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for hOAT with 14 will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.

Original languageEnglish (US)
Pages (from-to)8193-8207
Number of pages15
JournalJournal of the American Chemical Society
Issue number21
StatePublished - Jun 2 2021

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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