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

Journal Article · 06 September 2017 · Biochemistry

DOI: https://doi.org/10.1021/acs.biochem.7b00499 · OSTI ID:1438910

Mascarenhas, Romila ^[1]; Le, Hoang V. ^[2]; Clevenger, Kenneth D. ^[3]; Lehrer, Helaina J. ^[4]; Ringe, Dagmar ^[4]; Kelleher, Neil L.  ^[3]; Silverman, Richard B.  ^[3]; Liu, Dali  ^[1]

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1. Loyola Univ., Chicago, IL (United States)
2. Northwestern Univ., Evanston, IL (United States); Univ. of Mississippi, University, MS (United States)
3. Northwestern Univ., Evanston, IL (United States)
4. Brandeis Univ., Waltham, MA (United States)

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. Interestingly, 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.

• Research Organization: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22); Arthur J. Schmitt Foundation; Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor
• Grant/Contract Number: AC02-06CH11357
• OSTI ID: 1438910
• Journal Information: Biochemistry, Journal Name: Biochemistry Journal Issue: 37 Vol. 56; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)Copyright Statement
• Country of Publication: United States
• Language: ENGLISH