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

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 (3,4)-3-amino-4-fluorocyclopentenecarboxylic acid () 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 as an inactivator of human OAT. We have found that exhibits 10-fold less inactivation efficiency (/) against OAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with OAT. p and electrostatic potential calculations were performed to further support the notion that the α,β-unsaturated alkene of is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of over the corresponding saturated analogue (). Intact protein mass spectrometry and the crystal structure complex with OAT provide evidence to conclude that mainly inactivates OAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with OAT 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 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound exhibits unusual structural conversions which are catalyzed by specific residues within OAT, ultimately leading to an enamine mechanism-based inactivation of OAT through noncovalent interactions and covalent modification.