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The energy cost of urea synthesis
198 NH~- + CO2 + Aspartate + 3ATP + 2H20---* Urea + Fumarate + 2ADP + AMP + 2Pi + PPi + H + (1) The synthesis of urea is therefore considered to be an energy consuming process, the formation of each molecule leading to the loss of four high energy bonds. I-m This fact has been highlighted in many text books of biochemistry. While one author 4 comments that the cost of this form of detoxication of ammonia is surprisingly high, another has calculated that, for the privilege of excreting urea instead of ammonia, ureotelic animals lose about 15 per cent of the energy of the amino acids from which it is derived. 2
Some of the participants energetics of the Placenta); Jaime Mas-Oliva (Cholesterol transfer Proteins in Membranes).
Evaluation During the evaluation of the event in the form of a written report from those attending, the organizers noticed great enthusiasm, and their comments suggested that the activity had had a great impact. Next year, a follow-up program with 40 selected biochemistry teachers, will permit further evaluation of the impact of this kind of strategy in the quality of education of biochemistry at the different Schools of Medicine from the National University of Mexico.
Metabolic Aspects There are several aspects in the synthesis of urea which should be taken into account apart from the one represented by the overall equation. The following four aspects are closely related to the central pathway and the energy considerations involved in these should be taken into account before any conclusions are drawn: (1) Removal of the nitrogen atoms of amino acids and their conversion into ammonia. (2) Regeneration of aspartate by interaction with the enzymes of the Citrate Cylce. (3) Compartmentation of the enzymes of the urea cycle and transport of metabolites between the mitochondria and cytosol, and (4) Fate of the proton which is formed in the course of the synthesis of urea.
The Energy Cost of Urea Synthesis USHA ANAND and C V ANAND
Department of Biochemistry M S Ramaiah Medical College Bangalore 560 054, India
(1) Removal of the nitrogen of amino acids and its conversion to ammonia Amino acid I + c~-Ketoglutarate
transaminase
cx-Keto acid I + Glutamate
Introduction The u r e a cycle is a very important pathway which describes the mechanism by which the 'unwanted nitrogen' of amino acids is eliminated from the body. The entire sequence of reactions, which occurs in the mammalian liver, serves the vital function of disposal of ammonia, which is potentially toxic. One of the nitrogen atoms which is incorporated into urea is obtained from ammonia. The other is obtained from the amino group of aspartate. The carbon atom is obtained from respiratory carbon dioxide. ATP Requirement The overall process of synthesis of each molecule of urea, requires the utilization of three molecules of ATP, two of which are converted into A D P and Pi and one into AMP and PPi. Each turn of the cycle also leads to the consumption of 1 molecule of aspartate which is released as fumarate. The stoichiometric equation for the synthesis of urea can be represented as follows: BIOCHEMICAL
E D U C A T I O N 21(4) 1993
Glutamate + N A D +
(2)
Glutamate , ~-Ketoglutarate dehydrogenase
+ N A D H + H + + NH~- (3) The glutamate dehydrogenase reaction takes place in the mitochrondrial matrix and therefore the reduced N A D + can account for the production of 3 mol ATP. Transaminases are present both in the mitochondria and the cytosol and therefore reaction (3) may also be considered as taking place within the mitochondria. (2) The regeneration of aspartate The utilization of aspartate occurs in the cytosolic fraction of the cell and leads to the release of the carbon skeleton as fumarate. The regeneration of aspartate takes place by the interaction between the Citrate Cycle and the Urea Cycle using the following sequence of reactions: Fumarate + H 2 0 Fumarase Malate
(4)
199 Malate + NAD + MalateDH Oxaloacetate + NADH + H +
(5) Oxaloacetate + Glutamate
Transaminase
Aspartate + a-Ketoglutarate
(6)
The reoxidation of reduced NAD ÷ formed by malate dehydrogenase may also bring about the synthesis of 3 mol ATP (as in the case of the cytosolic enzyme of the glycolytic pathway namely glyceraldehyde 3-phosphate dehydrogenase). The ct-keto glutarate which is formed in reaction (6) can now undergo a transamination reaction with another amino acid (amino acid II) to regenerate glutamate. ct-Ketoglutarate + Amino acid II Transamin~e Glutamate + a-Keto acid II
(7)
Therefore even prior to the entry of the 2 nitrogen atoms into the cyclical pathway of urea synthesis there occur reactions accompanied by the generation of reducing equivalents. The formation of ammonia by glutamate dehydrogenase accounts for one while the sequences of fumarate, malate, oxaloacetate, aspartate accounts for the second one. (3) Compartmentation of the enzymes of the urea cycle and the transport of metabolites between them Among the enzymes that participate in the urea cycle carbamoyl phosphate synthetase and ornithine transcarbamylase are found within the mitochondria, while the others, argininosuccinate synthetase, argininosuccinase and arginase, are found in the cytosol. Once citrulline is formed by transcarbamylation from ornithine it has to be transported out of the mitochondria. This is said to occur by an equivalent inward transport of ornithine from the cystosol into the mitochondria. This transport mechanism is dependent upon the expenditure of energy. 5"1°The actual amount of energy that is utilized for this has been estimated to be equivalent to 0.25 pyrophosphate bonds. 5 For every turn of the urea cycle therefore the energy requirement is slightly more than 4 high-energy bonds (ie 4.25). The compartmentation of urea cycle enzymes is said to be necessary taking into account the concentration of the various metabolites involved in the process and their interaction with the Citrate Cycle. Particularly, the high concentration of fumarate within the mitochondria would inhibit the argininosuccinase reaction if it were to have an intramitochondrial location. The Keq of argininosuccinase reaction is 10-2M and outside the mitochondria the concentration of fumarate is not high enough to inhibit the splitting of argininosuccinate.l° (4) Fate of the proton which is produced in the course of urea synthesis According to (1) a proton is produced for
B I O C H E M I C A L EDUCATION 21(4) 1993
every molecule of urea that is synthesized. If this were to accumulate it would produce an acid load and lead to a disturbance in the acid base balance. However, under normal conditions, if the carbon skeleton of the amino acid is oxidized it produces a bicarbonate ion.it Generation of H ÷ by ureagenesis therefore is balanced by the HCO3- generated by the oxidation of amino acids.12 Only under acidotic conditions is there a depression of ureagenesis. The NH4- ion is eliminated by the kidney in exchange for a sodium ion. This exchange process in the renal tubules occurs at the expense of energy.
Total Energy Cost The energetics of urea synthesis under normal conditions could therefore be summarized as follows: The net energy gain in high energy bonds is +6, made up of glutamate DH (+3) and malate DH (+3), to be balanced against net energy utilization of -4.25 made up of carbamylphosphate synthetase ( - 2 ) , argininosuccinate synthetase ( - 2 ) and the ornithine-citrulline transport system (-025). Metabolic pathways do not function in isolation and in order to reach any clear consensus as to whether any pathway is consumptive of energy or not its interaction with other pathways should be considered. That there is a close interrelationship between the Urea Cycle and the Citrate Cycle has been discussed in several text books. 1-1o While the conversion of fumarate to aspartate as shown in eqn (4-6) has been dealt with in most of these, the energy gain has not been taken into account. So also the energy gain from the glutamate dehydrogenase reaction is not included for calculation of energetics. Since urea synthesis is meant for the disposal of unwanted nitrogen the process begins with the separation of nitrogen from the amino acids. The energy gain from this process therefore has to be taken into account. Urea synthesis should therefore not be considered as a costly process but as a self-sustaining one - - while one sequence of reactions utilizes energy it is more than compensated for by another sequence which produces it.
References i Stryer, L (1975) Biochemistry, Wiley, New York 2Lehninger, A L (1984) Principles of Biochemistry, Worth, New York 3Smith, E L and Others (1983) Principles of Biochemistry, Wiley, New York 4Zubay, G (1988) Biochemistry, Macmillan, New York 5McGilvery, R W (1983) Biochemistry: A Functional Approach, Saunders, Philadelphia ~Harper's Biochemistry (1990), Prentice Hall, Norwalk 7Bhagvan, N V (1980) Biochemistry, Jones and Bartlett, Boston ~Bohinski, R C (1979) Modern Concepts in Biochemistry, Allyn and Bacon, Boston ~Devlin, T M (1992) Textbook of Biochemistry with Clinical Correlations, Wiley, New York ~°Ottaway, J H and Apps, D K (1984) Biochemistry, 11Atkinson, D E and Bourke, E (1984) Trends Biochem Sci 9, 297-300 J2McCorquodale, D J (1992) Biochem Educ 20, 19-22
Some of the participants energetics of the Placenta); Jaime Mas-Oliva (Cholesterol transfer Proteins in Membranes).
Evaluation During the evaluation of the event in the form of a written report from those attending, the organizers noticed great enthusiasm, and their comments suggested that the activity had had a great impact. Next year, a follow-up program with 40 selected biochemistry teachers, will permit further evaluation of the impact of this kind of strategy in the quality of education of biochemistry at the different Schools of Medicine from the National University of Mexico.
Metabolic Aspects There are several aspects in the synthesis of urea which should be taken into account apart from the one represented by the overall equation. The following four aspects are closely related to the central pathway and the energy considerations involved in these should be taken into account before any conclusions are drawn: (1) Removal of the nitrogen atoms of amino acids and their conversion into ammonia. (2) Regeneration of aspartate by interaction with the enzymes of the Citrate Cylce. (3) Compartmentation of the enzymes of the urea cycle and transport of metabolites between the mitochondria and cytosol, and (4) Fate of the proton which is formed in the course of the synthesis of urea.
The Energy Cost of Urea Synthesis USHA ANAND and C V ANAND
Department of Biochemistry M S Ramaiah Medical College Bangalore 560 054, India
(1) Removal of the nitrogen of amino acids and its conversion to ammonia Amino acid I + c~-Ketoglutarate
transaminase
cx-Keto acid I + Glutamate
Introduction The u r e a cycle is a very important pathway which describes the mechanism by which the 'unwanted nitrogen' of amino acids is eliminated from the body. The entire sequence of reactions, which occurs in the mammalian liver, serves the vital function of disposal of ammonia, which is potentially toxic. One of the nitrogen atoms which is incorporated into urea is obtained from ammonia. The other is obtained from the amino group of aspartate. The carbon atom is obtained from respiratory carbon dioxide. ATP Requirement The overall process of synthesis of each molecule of urea, requires the utilization of three molecules of ATP, two of which are converted into A D P and Pi and one into AMP and PPi. Each turn of the cycle also leads to the consumption of 1 molecule of aspartate which is released as fumarate. The stoichiometric equation for the synthesis of urea can be represented as follows: BIOCHEMICAL
E D U C A T I O N 21(4) 1993
Glutamate + N A D +
(2)
Glutamate , ~-Ketoglutarate dehydrogenase
+ N A D H + H + + NH~- (3) The glutamate dehydrogenase reaction takes place in the mitochrondrial matrix and therefore the reduced N A D + can account for the production of 3 mol ATP. Transaminases are present both in the mitochondria and the cytosol and therefore reaction (3) may also be considered as taking place within the mitochondria. (2) The regeneration of aspartate The utilization of aspartate occurs in the cytosolic fraction of the cell and leads to the release of the carbon skeleton as fumarate. The regeneration of aspartate takes place by the interaction between the Citrate Cycle and the Urea Cycle using the following sequence of reactions: Fumarate + H 2 0 Fumarase Malate
(4)
199 Malate + NAD + MalateDH Oxaloacetate + NADH + H +
(5) Oxaloacetate + Glutamate
Transaminase
Aspartate + a-Ketoglutarate
(6)
The reoxidation of reduced NAD ÷ formed by malate dehydrogenase may also bring about the synthesis of 3 mol ATP (as in the case of the cytosolic enzyme of the glycolytic pathway namely glyceraldehyde 3-phosphate dehydrogenase). The ct-keto glutarate which is formed in reaction (6) can now undergo a transamination reaction with another amino acid (amino acid II) to regenerate glutamate. ct-Ketoglutarate + Amino acid II Transamin~e Glutamate + a-Keto acid II
(7)
Therefore even prior to the entry of the 2 nitrogen atoms into the cyclical pathway of urea synthesis there occur reactions accompanied by the generation of reducing equivalents. The formation of ammonia by glutamate dehydrogenase accounts for one while the sequences of fumarate, malate, oxaloacetate, aspartate accounts for the second one. (3) Compartmentation of the enzymes of the urea cycle and the transport of metabolites between them Among the enzymes that participate in the urea cycle carbamoyl phosphate synthetase and ornithine transcarbamylase are found within the mitochondria, while the others, argininosuccinate synthetase, argininosuccinase and arginase, are found in the cytosol. Once citrulline is formed by transcarbamylation from ornithine it has to be transported out of the mitochondria. This is said to occur by an equivalent inward transport of ornithine from the cystosol into the mitochondria. This transport mechanism is dependent upon the expenditure of energy. 5"1°The actual amount of energy that is utilized for this has been estimated to be equivalent to 0.25 pyrophosphate bonds. 5 For every turn of the urea cycle therefore the energy requirement is slightly more than 4 high-energy bonds (ie 4.25). The compartmentation of urea cycle enzymes is said to be necessary taking into account the concentration of the various metabolites involved in the process and their interaction with the Citrate Cycle. Particularly, the high concentration of fumarate within the mitochondria would inhibit the argininosuccinase reaction if it were to have an intramitochondrial location. The Keq of argininosuccinase reaction is 10-2M and outside the mitochondria the concentration of fumarate is not high enough to inhibit the splitting of argininosuccinate.l° (4) Fate of the proton which is produced in the course of urea synthesis According to (1) a proton is produced for
B I O C H E M I C A L EDUCATION 21(4) 1993
every molecule of urea that is synthesized. If this were to accumulate it would produce an acid load and lead to a disturbance in the acid base balance. However, under normal conditions, if the carbon skeleton of the amino acid is oxidized it produces a bicarbonate ion.it Generation of H ÷ by ureagenesis therefore is balanced by the HCO3- generated by the oxidation of amino acids.12 Only under acidotic conditions is there a depression of ureagenesis. The NH4- ion is eliminated by the kidney in exchange for a sodium ion. This exchange process in the renal tubules occurs at the expense of energy.
Total Energy Cost The energetics of urea synthesis under normal conditions could therefore be summarized as follows: The net energy gain in high energy bonds is +6, made up of glutamate DH (+3) and malate DH (+3), to be balanced against net energy utilization of -4.25 made up of carbamylphosphate synthetase ( - 2 ) , argininosuccinate synthetase ( - 2 ) and the ornithine-citrulline transport system (-025). Metabolic pathways do not function in isolation and in order to reach any clear consensus as to whether any pathway is consumptive of energy or not its interaction with other pathways should be considered. That there is a close interrelationship between the Urea Cycle and the Citrate Cycle has been discussed in several text books. 1-1o While the conversion of fumarate to aspartate as shown in eqn (4-6) has been dealt with in most of these, the energy gain has not been taken into account. So also the energy gain from the glutamate dehydrogenase reaction is not included for calculation of energetics. Since urea synthesis is meant for the disposal of unwanted nitrogen the process begins with the separation of nitrogen from the amino acids. The energy gain from this process therefore has to be taken into account. Urea synthesis should therefore not be considered as a costly process but as a self-sustaining one - - while one sequence of reactions utilizes energy it is more than compensated for by another sequence which produces it.
References i Stryer, L (1975) Biochemistry, Wiley, New York 2Lehninger, A L (1984) Principles of Biochemistry, Worth, New York 3Smith, E L and Others (1983) Principles of Biochemistry, Wiley, New York 4Zubay, G (1988) Biochemistry, Macmillan, New York 5McGilvery, R W (1983) Biochemistry: A Functional Approach, Saunders, Philadelphia ~Harper's Biochemistry (1990), Prentice Hall, Norwalk 7Bhagvan, N V (1980) Biochemistry, Jones and Bartlett, Boston ~Bohinski, R C (1979) Modern Concepts in Biochemistry, Allyn and Bacon, Boston ~Devlin, T M (1992) Textbook of Biochemistry with Clinical Correlations, Wiley, New York ~°Ottaway, J H and Apps, D K (1984) Biochemistry, 11Atkinson, D E and Bourke, E (1984) Trends Biochem Sci 9, 297-300 J2McCorquodale, D J (1992) Biochem Educ 20, 19-22