In vivo inactivation of formylglycinamidine ribonucleotide synthetase in rat hepatoma

Abstract

The antitumor drug acivicin, l -(αS,5S)-α- amino-3- chloro-4,5- dihydro-5- isoxazoleacetic acid, irreversibly inactivated in vivo formylglycinamidine ribonucleotide synthetase (FGAM synthetase, EC 6.3.5.3) in transplantable rat hepatoma 3924A while the activity in host liver remained unchanged. At acivicin doses of 1.0 and 5.0 mg/kg body weight, enzyme activity in the hepatoma decreased to 26 and 5%, respectively, after 2 hr. The activity of the in vivo inactivated hepatoma 3924A enzyme could not be restored by gel filtration or 40 hr of dialysis. In the absence of l-glutamine, acivicin in vitro inactivated both liver and hepatoma FGAM synthetase in a time-dependent fashion, with an inactivation constant K inact = 66 μM and a minimum inactivation half-time T = 1.0 min. In the presence of l-glutamine, competitive inhibition was observed with a K i = 5 μM. Protection against in vitro inactivation was observed in the presence of 1 mM l-glutamine, suggesting that l-glutamine concentrations are important in the selective toxicity of acivicin on hepatoma cells in vivo. Irreversible inhibition of FGAM synthetase by acivicin is consistent with the view that this antibiotic is an active site-directed affinity analog of l-glutamine and indicates that this enzyme is a sensitive target of acivicin action.