Rutgers University, New Brunswick, New Jersey, USA
The ornithine cycle, also known as the urea cycle and the Krebs-Henseleit cycle, is the pathway in mammalian liver that allows the detoxification and excretion of excess nitrogen as urea. Flux through the cycle is driven by the demand to remove excess ammonia derived from the degradation of amino acids that arise either from the diet or from endogenous proteolysis. In a healthy individual, consuming a typical western diet, flux through the ornithine cycle produces some 30-35 g of urea per day but as much as 25% of this can be recycled via hydrolysis to ammonia and bicarbonate by colonic bacteria.
Three major systems have evolved for the elimination of excess nitrogen from the body, and animals may be classified according to their major nitrogenous excretion product. Thus most fish are ammoniatelic (ammonia excretion), reptiles and birds are uricotelic (uric acid excretion), and mammals are ureotelic (urea excretion). In addition, urea excretion is seen in some teleost fish, and urea synthesis is also present in elasmobranchs (sharks and rays) where urea functions primarily in an osmotic role, not in nitrogen excretion.
Urea was first described as "substance savoneuse," that yielded ammonia on hydrolysis, isolated from urine by Roulle in 1773 and was obtained in pure form by Proust in 1820. There is evidence that Davy synthesized urea from cabonyl chloride and ammonia in 1812 but he failed to recognize the product. Thus Wholer is credited for achieving the synthesis, in 1828, of urea from ammonia and lead cyanate in what is best remembered as the first synthesis of an organic compound from inorganic substrates. Over the next century many theories were developed to explain urea synthesis but the true pathway was not discovered until 1932. Working with liver slices in incubation Krebs and Henseleit found that the addition of small amounts of ornithine greatly accelerated the synthesis of urea from ammonia and furthermore, that the ornithine could be recovered at the end of the incubation. From this they formulated the idea that ornithine was acting as a "catalyst." They also identified citrulline as an intermediate and described the ornithine cycle as ammonia (together with bicarbonate) and ornithine combining to give citrulline that was then converted to arginine. The final step was hydrolysis of the arginine to urea and with the concomitant regeneration of ornithine. This was the first description of a cyclic biochemical pathway again illustrating the central role urea synthesis has had in the history of biology and chemistry.
Since Krebs and Henseleit's original discovery, the chemistry of the cycle has been completely described and although the ornithine cycle consists only of four enzymes, the need for carbamoyl phosphate means that carbamoyl phosphate synthetase 1 is usually considered an ornithine cycle enzyme.
The complete cycle (Figure 1) is only expressed in liver parenchymal cells with highest activity in those cells near the portal inlet (periportal cells). The substrates for the cycle are ammonia, bicarbonate, aspartate, and three ATP equivalents. In this text the term ammonia refers to the sum of NH4 plus NH3, and where a specific molecular species is important, it is shown in parentheses. Beginning in the mitochondria ammonia (NH3) combines with bicarbonate to form carbamoyl phosphate involving 2 ATP and releasing 2 ADP plus inorganic phosphate, through the action of carbamoyl phosphate synthetase 1 (EC 22.214.171.124). The carbamoyl group is then transferred to ornithine via ornithine transcarbamoylase (EC 126.96.36.199) to yield citrulline that leaves the mitochondria in exchange for ornithine on the ORN 1 transporter. In the cytosol, citrulline condenses with aspartate in a reaction, requiring ATP and the formation of a citrulline-AMP intermediate, to give argininosuccinate, AMP, and pyrophosphate via the action of argininosuccinate synthetase (EC 188.8.131.52). The carbon skeleton of the aspartate is then cleaved from argininosuccinate by argininosuccinate lyase (EC 184.108.40.206) to yield fumarate and arginine. The final step is hydrolysis of arginine by arginase (EC 220.127.116.11) to give the end product urea with the regeneration of ornithine that can enter the mitochondria on the ORN 1 transporter and begin another round of the cycle.
Channeling in the Cycle
Although carbamoyl phosphate synthetase 1 and ornithine transcarbamoylase are mitochondrial matrix enzymes, they are localized in very close proximity to the cristae of the inner membrane. This would favor direct transfer of intermediates along the pathway and tracer experiments have shown that extra-mitochondrial ornithine is preferentially utilized for citrulline synthesis over ornithine generated within the mitochondria. Such findings indicate that the ORN 1 transporter, carbamoyl phosphate synthetase 1 and ornithine transcarbamoylase are working efficiently as a unit without free mixing of the intermediates within the mitochondria. In addition, there is evidence of similar channeling within the cytosolic section of the cycle. In permeabilized hepato-cytes incubated with 14C labeled bicarbonate, the formation of labeled urea is not significantly diluted by the addition of large amounts of cycle intermediates
(arginine, argininosuccinate, or citrulline), again showing no mixing with exogenous intermediates. Therefore all intermediates of the cycle appear to be carefully channeled along the pathway which may be related to the need to ensure removal of the highly toxic substrate, ammonia.
The function of the ornithine cycle is to detoxify excess ammonia in the mammalian body and as such the source of the ammonia must be considered whenever the cycle is discussed in more than simple chemical terms. There are two types of condition when the delivery of ammonia within the liver is high and thus require high rates of urea synthesis. The first is in response to dietary (exogenous) protein when excess amino acids are degraded and the second is in response to increased catabolism of endogenous amino acids, arising from proteolysis, during early starvation and in hypercata-bolic states such as those that arise from sepsis and injury. A quantitative analysis of the fate of a typical daily load of dietary amino acids showed that, if those amino acids that must be catabolized within the liver were to undergo complete oxidation then, this would produce more ATP than the liver uses in a day. Since the liver oxidizes other fuels in addition to amino acids, it is evident that the carbon skeletons of the amino acids cannot undergo complete oxidation in the liver. In practice the amino acid carbon skeletons are conserved as either glycogen (indirect pathway of synthesis) or released into the circulation as free glucose. In the case of starvation and hypercatabolic states, the reason the amino acids are being degraded is to provide glucose for the body, and urea synthesis is simply coincidental. Thus the synthesis of urea from amino acids is always linked to the hepatic pathway of gluconeogenesis.
There is currently some debate about how much of the ammonia and aspartate for the cycle are taken up from the circulation directly and how much are synthesized within the liver from ammonia and/or other amino acids. Within the liver however, glutamate dehydrogenase and the major aminotransferases catalyze reactions close to equilibrium which allows the maintenance of a balanced substrate supply to the cycle (Figure 2).
By following the fate of a single amino acid, alanine, through hepatic metabolism, the integrative nature of the ornithine cycle with gluconeogenesis can be seen (Figure 3). Alanine undergoes transamination with a-ketoglutarate in the cytosol and both the pyruvate and glutamate formed enter the mitochondria where the pyruvate is carboxylated to oxaloacetate via the action of pyruvate carboxylase. The glutamate then transaminates with this oxaloacetate to give aspartate and a-ketoglutarate. The latter can then recycle via the a-Amino acid
Transaminase a-Keto acid
Glutamat e dehydrogenase
FIGURE 2 The equilibrium nature of glutamate dehydrogenase and the transaminases. Amino acids can transfer their amino group to a-ketoglutarate to produce glutamate via transamination. Glutamate can yield ammonia and a-ketoglutarate through glutamate dehydrogenase. Since glutamate dehydrogenase and the major transminases catalyze reactions close to equilibrium the entire system is freely reversible and thus ensures a balance of substrates, NH3 and aspartate (produced through aspartate aminotransferase) for the ornithine cycle.
cytosol to transaminate with another alanine. The aspartate leaves the mitochondria to be incorporated into the cycle by argininosuccinate synthetase in the cytosol. At the subsequent lyase step the carbon skeleton released as fumarate. Since fumarate is unable to is
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