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Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5′-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover

Mascarenhas, Romila, Le, Hoang V., Clevenger, Kenneth D., Lehrer, Helaina J., Ringe, Dagmar, Kelleher, Neil L., Silverman, Richard B., Liu, Dali
Biochemistry 2017 v.56 no.37 pp. 4951-4961
aspartate transaminase, binding sites, crystal structure, drugs, gamma-aminobutyric acid, hepatoma, mass spectrometry, ornithine-oxo-acid transaminase, pyridoxal, pyridoxal phosphate
Potent mechanism-based inactivators can be rationally designed against pyridoxal 5′-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.