In vivo inactivation by acivicin of carbamoyl-phosphate synthetase II in rat hepatoma.

The antitumor drug acivicin, L-(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, in vivo irreversibly inactivated carbamoyl-phosphate synthetase II(glutamine-dependent)(EC 6.3.5.5), the first and rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, in transplantable rat hepatoma and host liver. With two injections of 0.5 mg acivicin per 100 g body weight to one group and two injections of 5 mg to another group, enzyme activity decreased to 20 and 1% in hepatoma and to 99 and 31% in liver respectively. Aspartate carbamoyltransferase (EC 2.1.3.2) activity was not affected. Acivicin in vitro selectively inactivated glutamine-dependent activity of the synthetase II from the hepatoma and liver, with an inactivation constant (Kinact) of 90 microM and a minimum inactivation half-time (T) of 0.7 min. The inactivation velocity with 10 microM acivicin was 5.0-fold stimulated by 2 mM MgATP and 18.4-fold by 2 mM MgATP plus 16.7 mM bicarbonate. MgATP at 0.5 mM caused half-maximum stimulation of the inactivation velocity. Under in vitro conditions, L-glutamine (1 mM) protected the enzyme from inactivation by 10 microM acivicin. The synthetase activity was protected in vitro by 6 mM concentrations for glycine (84%), L-glutamate (59%) and L-aspartate (51%) and by 0.5 mM UTP (35%) from inactivation by 20 microM acivicin. The results are compatible with the suggestion that acivicin is an active site-directed affinity analog of L-glutamine.