The inhibition of urea biosynthesis by analogues of aspartic acid was studied in vitro in homogenates and enzyme preparations from rat liver. Each of the analogues tested inhibited the overall utilization of citrulline for urea formation by liver homogenates. The concentrations required to give 50% inhibition were: N-allylaspartate, 0.248 M; α-methylaspartate, 0.140 M; β-methylaspartate, 0.078 M; and β-hydroxy-β-methylaspartate, 0.038 M. The β-substituted analogues partly replaced aspartate as a substrate for citrulline utilization in liver homogenates. The replacement was probably due to transamination of the analogues with oxaloacetate, since the effect was not observed when the assay mixture did not contain a substrate which could yield oxaloacetate.A study of individual enzymes of the urea cycle showed that arginase, argininosuccinase, and ornithine transcarbamylase were not greatly affected by the analogues. However, carbamyl phosphate synthetase as well as argininosuccinate synthetase were strongly inhibited, suggesting that the analogues act by some mechanism other than simple antagonism of aspartate. Part of the inhibition was related to the ability of the analogues to complex Mg2+, since increased concentrations of Mg2+ prevented the inhibition of carbamyl phosphate synthetase and reduced the inhibition of argininosuccinate synthetase by α-methylaspartate and N-allylaspartate. In addition, β-methylaspartate was found to depress oxidative and phosphorylative reactions, thus interfering with the energy production required for urea formation.Aspartic acid in concentrations comparable with those required to effect inhibition by α-methylaspartate produced a marked inhibition of citrulline utilization in liver homogenates and of purified argininosuccinate synthetase. This observation suggests that part of the inhibitions observed with the analogues are of the "substrate type".
In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.
