EFFECT OF THE TRANSITION-STATE ANALOG, DELTA-N-(PHOSPHONACETYL)-L-ORNITHINE ON CITRULLINE SYNTHESIS IN ISOLATED RAT-LIVER MITOCHONDRIA AND ON UREA SYNTHESIS IN ISOLATED RAT HEPATOCYTES

δ‐N‐(Phosphonacetyl)‐l‐ornithine [Orn(PAc)], a transition state analogue for ornithine transcarbamylase, is an effective inhibitor of citrulline synthesis in isolated rat liver mitochondria, and urea synthesis in isolated hepatocytes. In intact mitochondria, the efficacy of inhibition of citrulline synthesis by Orn(PAc) was shown to be dependent on the conditions of incubation. The citrulline biosynthetic capacity of intact mitochondria was reduced to 60% of controls by as little as 0.01 mM inhibitor, and to 20% of controls by 0.05 mM inhibitor in mitochondria treated with 0.5% Triton X‐100. The greater capacity of Orn(PAc) to inhibit citrulline synthesis after treatment of mitochondria with Triton X‐100 was in part due to the elimination of a permeability barrier to reactants of the carbamoyl‐phosphate synthetase (ammonia) reaction. Thus, maximum inhibition by Orn(PAc) was dependent on the relative activities of carbamoyl‐phosphate synthetase (ammonia) and ornithine transcarbamylase, and inhibition could be completely reversed by stimulating ornithine transcarbamylase using ornithine. When added to isolated hepatocytes, Orn(PAc) inhibited urea biosynthesis by up to 20% at 0.5 mM concentration of inhibitor. This inhibition in the conversion of carbamoyl phosphate to citrulline had a marked effect on the utilization of intra‐mitochondrially synthesized carbamoyl phosphate for extramitochondrial synthesis of pyrimidines. There was up to a twofold stimulation in the rate of synthesis of orotate and uridine nucleotides when Orn(PAc) was added to hepatocytes incubated in basal medium (Eagles) supplemented with 10 mM NH4Cl. These data confirm clinical observations that one of the consequences of a reduction in ornithine transcarbamylase activity, such as occurs in certain disease states, is a stimulation in pyrimidine biosynthesis, and further suggests that transition‐state analogues, such as Orn(PAc), may be useful in metabolic modelling studies on the effects of enzymic defects in mammalian tissues. Copyright © 1979, Wiley Blackwell. All rights reserved