Copyright © 2000-2003 Mark Brandt, Ph.D. 46The Urea CycleAs has been mentioned, ammonium is toxic, and even small amounts will damagethe nervous system. Genetic disorders in ammonium metabolism result inavoidance of high-protein foods and in mental retardation. Ammonium intoxication(e.g., as a result of decreased liver function) can be lethal. As a result, animals mustcontrol the amounts of free ammonium that are present, and often use a formorganic nitrogen as a waste product. In humans and most other terrestrialmammals, urea is the major nitrogen excretion product. Urea has the advantagesof being relatively inexpensive to produce, being soluble in water, and being non-toxic.Although most tissues can synthesize urea, most urea is produced in the liver.Because urea is uncharged, urea excretion does not involve the loss of anyelectrolytes as counter ions. Excretion of urea is, however, associated withconsiderable loss of water due to osmotic pressure.Urea is produced as part of the series of reactions that comprise the urea cycle. Theurea cycle is the first of the two major metabolic cycles discovered by Hans Krebs.In fact, the urea cycle was the first biological cycle to be discovered, and helpedestablish the concept for the discovery of the TCA cycle.Copyright © 2000-2003 Mark Brandt, Ph.D. 47The urea cycle is smaller than the TCA cycle, and has fewer intermediates.Interestingly, all of the four intermediates are a-amino acids, although three,ornithine, citrulline, and argininosuccinate are not found in proteins.The urea cycle requires five reactions (of which four are part of the actual cycle).The first reaction is the primary regulated step. Carbamoyl phosphatesynthetase I5 is the mitochondrial enzyme that catalyzes the formation ofcarbamoyl phosphate from inorganic ammonium and carbonate. This enzyme isthus another enzyme capable of fixing ammonium. The usual fate of the ammoniumfixed by carbamoyl phosphate synthetase I is excretion in the form of urea, andtherefore this enzyme is usually considered separately from glutamine synthetaseand glutamate dehydrogenase, which fix ammonium for use in metabolism.In eukaryotic organisms, a different carbamoyl phosphate synthetase formscarbamoyl phosphate in the cytoplasm as the first step in pyrimidine biosynthesis.Unlike carbamoyl phosphate synthetase I, however, carbamoyl phosphatesynthetase II uses glutamine as the ammonium donor instead of free ammonium.Carbamoyl phosphate synthetase I requires the presence of the allosteric activatorN-acetylglutamate (the product of the first step in ornithine biosynthesis) foractivity. This regulation means that carbamoyl phosphate synthetase I is therate-limiting enzyme of the urea cycle.The other four enzymes are part of the actual cycle. The cycle begins with theaddition of carbamoyl phosphate to ornithine by ornithine transcarbamoylase toproduce citrulline. Citrulline then leaves the mitochondria using a specifictransporter, because the remaining reactions occur in the cytoplasm. Once in thecytoplasm, citrulline is combined with aspartate by argininosuccinatesynthetase to form argininosuccinate, in a reaction that requires ATP, andproduces AMP and pyrophosphate. The next enzyme, argininosuccinase,performs a cleavage reaction that releases the TCA cycle intermediate fumarateand the amino acid arginine. Note that the arginine contains nitrogens derived fromornithine, from the free ammonium, and from the aspartate. Arginine is thencleaved by arginase to release urea and to regenerate ornithine. Ornithine also hasa specific transporter that allows the ornithine to re-enter the mitochondria,completing the cycle.As with the TCA cycle, the urea cycle is controlled by two factors: regulatedenzymes and substrate availability. For the urea cycle the regulated enzyme iscarbamoyl phosphate synthetase I. For the urea cycle, the availability of cycleintermediates and free ammonium also control the cycle. Thus, high levels ofornithine allow the cycle to proceed more rapidly. 5 Some textbooks call this enzyme carbamoyl phosphate “synthase” rather than “synthetase”. Thestrict nomenclature rule states that a “synthetase” is an enzyme that combines two molecules usingATP to provide the driving force, while a “synthase” combines two molecules without using ATP. Forthe purpose of this course, “synthase” and “synthetase” are effectively used interchangeably,although I am attempting to eliminate inconsistent usage.Copyright © 2000-2003 Mark Brandt, Ph.D. 48In principle, the urea cycle can be used to synthesize or degrade arginine. Note,however that net synthesis of arginine requires input of one of the other urea cycleintermediates; net degradation of arginine requires net removal of one of theseintermediates. As described about, the urea cycle does not result in an alteration inthe amount of arginine.OrnithineOrnithine is the equivalent of the TCA cycle intermediate oxaloacetate; levels ofornithine tend to control the rate of the urea cycle. Ornithine can be produced inseveral ways. One method of increasing ornithine levels is to take up arginine froma source outside the cell (either from protein breakdown or from a dietary source). Asecond method is to synthesize ornithine directly. Ornithine synthesis normallybegins with glutamate, although proline can also act as a source of ornithinesynthesis.One pathway for the conversion of glutamate to ornithine is similar to the pathwayfor proline synthesis. However, the first step in the ornithine synthesis pathway,the N-acetylation of glutamate by N-acetylglutamate synthase forces ornithinerather than proline production. The N-acetyl group acts as a protecting group; lackof a free primary amine prevents the non-enzymatic pyrroline ring formation byglutamate-5-semialdehyde. N-acetylglutamate synthase also acts to produce the N-acetylglutamate required for carbamoyl phosphate synthetase I activity. The nexttwo reactions, the phosphorylation of N-acetylglutamate by ATP, and the NADPH-dependent dephosphosphorylation reaction use ATP and NADPH to drive theproduction of N-acetylglutamate-5-semialdehyde.