The Urea Cycle As has been mentioned ammonium is toxic and even small amounts will damage the nervous system Genetic disorders in ammonium metabolism result in avoidance 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 must control the amounts of free ammonium that are present and often use a form organic nitrogen as a waste product In humans and most other terrestrial mammals urea is the major nitrogen excretion product Urea has the advantages of being relatively inexpensive to produce being soluble in water and being nontoxic 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 any electrolytes as counter ions Excretion of urea is however associated with considerable loss of water due to osmotic pressure Urea is produced as part of the series of reactions that comprise the urea cycle The urea 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 helped establish the concept for the discovery of the TCA cycle Copyright 2000 2003 Mark Brandt Ph D 46 The 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 phosphate synthetase I5 is the mitochondrial enzyme that catalyzes the formation of carbamoyl phosphate from inorganic ammonium and carbonate This enzyme is thus another enzyme capable of fixing ammonium The usual fate of the ammonium fixed by carbamoyl phosphate synthetase I is excretion in the form of urea and therefore this enzyme is usually considered separately from glutamine synthetase and glutamate dehydrogenase which fix ammonium for use in metabolism In eukaryotic organisms a different carbamoyl phosphate synthetase forms carbamoyl phosphate in the cytoplasm as the first step in pyrimidine biosynthesis Unlike carbamoyl phosphate synthetase I however carbamoyl phosphate synthetase II uses glutamine as the ammonium donor instead of free ammonium Carbamoyl phosphate synthetase I requires the presence of the allosteric activator N acetylglutamate the product of the first step in ornithine biosynthesis for activity This regulation means that carbamoyl phosphate synthetase I is the rate limiting enzyme of the urea cycle The other four enzymes are part of the actual cycle The cycle begins with the addition of carbamoyl phosphate to ornithine by ornithine transcarbamoylase to produce citrulline Citrulline then leaves the mitochondria using a specific transporter because the remaining reactions occur in the cytoplasm Once in the cytoplasm citrulline is combined with aspartate by argininosuccinate synthetase to form argininosuccinate in a reaction that requires ATP and produces AMP and pyrophosphate The next enzyme argininosuccinase performs a cleavage reaction that releases the TCA cycle intermediate fumarate and the amino acid arginine Note that the arginine contains nitrogens derived from ornithine from the free ammonium and from the aspartate Arginine is then cleaved by arginase to release urea and to regenerate ornithine Ornithine also has a 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 regulated enzymes and substrate availability For the urea cycle the regulated enzyme is carbamoyl phosphate synthetase I For the urea cycle the availability of cycle intermediates and free ammonium also control the cycle Thus high levels of ornithine allow the cycle to proceed more rapidly 5 Some textbooks call this enzyme carbamoyl phosphate synthase rather than synthetase The strict nomenclature rule states that a synthetase is an enzyme that combines two molecules using ATP to provide the driving force while a synthase combines two molecules without using ATP For the 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 47 In 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 cycle intermediates net degradation of arginine requires net removal of one of these intermediates As described about the urea cycle does not result in an alteration in the amount of arginine Ornithine Ornithine is the equivalent of the TCA cycle intermediate oxaloacetate levels of ornithine tend to control the rate of the urea cycle Ornithine can be produced in several ways One method of increasing ornithine levels is to take up arginine from a source outside the cell either from protein breakdown or from a dietary source A second method is to synthesize ornithine directly Ornithine synthesis normally begins with glutamate although proline can also act as a source of ornithine synthesis One pathway for the conversion of glutamate to ornithine is similar to the pathway for proline synthesis However the first step in the ornithine synthesis pathway the N acetylation of glutamate by N acetylglutamate synthase forces ornithine rather than proline production The N acetyl group acts as a protecting group lack of a free primary amine prevents the non enzymatic pyrroline ring formation by glutamate 5 semialdehyde N acetylglutamate synthase also acts to produce the Nacetylglutamate required for carbamoyl phosphate synthetase I activity The next two reactions the phosphorylation of N acetylglutamate by ATP and the NADPHdependent dephosphosphorylation reaction use ATP and NADPH to drive the production of N acetylglutamate 5 semialdehyde The semialdehyde oxygen is then replaced with an amino group by N acetylornithine d aminotransferase followed by deprotection of the product by N acetylornithine deacetylase As with the kinase reaction the loss of the protecting acetyl group helps to make the pathway irreversible Ornithine can also be synthesized from unprotected glutamate 5 semialdehyde by ornithine d aminotransferase in a reverse of the ornithine breakdown pathway The Copyright 2000 2003 Mark Brandt Ph