An investigation of the properties of ornithine aminotransferase after inactivation by the ‘suicide’ inhibitor aminohexynoate and use of the compound as a probe of intracellullar protein turnover

Abstract

Ornithine aminotransferase is shown to bind 1 mol of amino[14C]hexynoate per mol of coenzyme in the ‘suicide’ inactivation process. At the same time the coenzyme pyridoxal phosphate becomes irreversibly bound to the enzyme protein. Apart from the inactivation, the labelled enzyme is indistinguishable from native ornithine aminotransferase by several separation techniques. Because the rate of degradation of the labelled enzyme is the same as that of the normal enzyme it is concluded that loss of coenzyme does not initiate turnover. Free aminohexynoate is rapidly eliminated from the liver, and 70% of the compound is excreted unchanged in 7.5 h. Inactivated ornithine aminotransferase accounts for 11% of the total labelled liver protein and significant amounts of label are found in aspartate aminotransferase which is also extensively inactivated. The rate of return of enzyme activity is determined and found to be more rapid than expected for a process in which the enzyme is synthesized at a constant rate and degraded in a single, first-order process.

dl-canaline and 5-fluoromethylornithine. Comparison of two inactivators of ornithine aminotransferase

“…The absorption spectrum of OAT that is generated gradually after inactivation with 5FMOrn (Fig. 4) is different from that obtained after inactivation with (R/S)-4-aminohex-5-ynoic acid (Jones et al, 1983). In the latter case the electron delocalization of the coenzyme is restricted to the pyridine ring, which results in an absorption maximum at 330 nm.…”

“…For (R/S)-4-aminohex-5-ynoic acid, an inactivator of both OAT and 4-aminobutyrate aminotransferase (EC 2.6.1.19) (Jung & Metcalf, 1975;Jung & Seiler, 1978), covalent binding to an amino acid residue within the active site of OAT was demonstrated (Jones et al, 1983). That 5FMOrn, like ornithine, reacts first with pyridoxal 5'-phosphate is clear from the initial spectral changes and also from the fact that OAT is protected from inactivation by L-ornithine (Daune et al, 1988).…”

5-Fluoromethylornithine, an irreversible and specific inhibitor of l-ornithine:2-oxo-acid aminotransferase

“…One may assume that the reactions involved in the inactivation of OAT by 5-FMOrn are analogous to those suggested by Bey et al (1981) for the inactivation of GABA-T by some w-fluoromethyl derivatives of ,J-alanine and 4-aminobutyrate, because OAT and GABA-T are enzymes with comparable mechanism (Williams et al, 1982;Metcalf, 1979). It is therefore not surprising that some inactivators of GABA-T are also potent inactivators of OAT Yasuda et al, 1980;Jones et al, 1983). 2-Fluoromethylornithine and 2-difluoromethylornit-hine are enzyme-activated irreversible inhibitors of ODC (Bey, 1978).…”

Ornithine Aminotransferase, an Important Glutamate-Metabolizing Enzyme at the Crossroads of Multiple Metabolic Pathways

“…The OAT degradation rate constant is around 0.4 day −1 in the liver [35,115,116], i.e. a half-life of about 1.7 days (but only 0.95 days in the rat [117]).…”

“…OAT is inhibited by aminohexanoate [35], GABA, gabaculine, 5-fluoromethylornithine (5-FMOrn) [20,36,37,38], and l -canaline [39]. A more complete list of inhibitors and their structures is given in Table 2.…”