1. Section 8. Amino Acid Metabolism Urea cycle 11/18/05

2. Substrates for the Urea Cycle Above, amino groups are transferred to glutamate, from which ammonium is produced, and then used to make carbamoyl phosphate.Above, amino groups are transferred to glutamate, from which ammonium is produced, and then used to make carbamoyl phosphate. Below, amino groups are transferred to produce aspartate.Below, amino groups are transferred to produce aspartate. Above, amino groups are transferred to glutamate, from which ammonium is produced, and then used to make carbamoyl phosphate.Above, amino groups are transferred to glutamate, from which ammonium is produced, and then used to make carbamoyl phosphate. Below, amino groups are transferred to produce aspartate.Below, amino groups are transferred to produce aspartate. 1

3. Urea Cycle Aspartate and carbamoyl phosphate each deliver an amino group to the cycle.Aspartate and carbamoyl phosphate each deliver an amino group to the cycle. Notice that the carbamoyl phosphate production and condensation occur in the mitochondrial matrix.Notice that the carbamoyl phosphate production and condensation occur in the mitochondrial matrix. Aspartate and carbamoyl phosphate each deliver an amino group to the cycle.Aspartate and carbamoyl phosphate each deliver an amino group to the cycle. Notice that the carbamoyl phosphate production and condensation occur in the mitochondrial matrix.Notice that the carbamoyl phosphate production and condensation occur in the mitochondrial matrix. 2 Fig. 23.16

4. NH 4 + from Oxidative Deamination of Glutamate Hexameric glutamate dehydrogenase is is controlled allosterically.Hexameric glutamate dehydrogenase is is controlled allosterically. –High energy levels inhibit (ATP and GTP). –Low energy levels activate (ADP and GDP). NADP + can replace NAD +.NADP + can replace NAD +. NH 4 +, which is toxic to humans, is produced in the mitochondria and used to make carbamoyl phosphate.NH 4 +, which is toxic to humans, is produced in the mitochondria and used to make carbamoyl phosphate. Hexameric glutamate dehydrogenase is is controlled allosterically.Hexameric glutamate dehydrogenase is is controlled allosterically. –High energy levels inhibit (ATP and GTP). –Low energy levels activate (ADP and GDP). NADP + can replace NAD +.NADP + can replace NAD +. NH 4 +, which is toxic to humans, is produced in the mitochondria and used to make carbamoyl phosphate.NH 4 +, which is toxic to humans, is produced in the mitochondria and used to make carbamoyl phosphate. 3

5. Carbamoyl Phosphate Synthesis Carbamoyl phosphate synthetase is in mitochondrial matrix.Carbamoyl phosphate synthetase is in mitochondrial matrix. NH 4 + is source of NH 3.NH 4 + is source of NH 3. The hydrolysis of two ATP make this reaction essentially irreversible.The hydrolysis of two ATP make this reaction essentially irreversible. N-acetyl glutamate is an allosteric activator. (see S08L05)N-acetyl glutamate is an allosteric activator. (see S08L05) Carbamoyl phosphate synthetase is in mitochondrial matrix.Carbamoyl phosphate synthetase is in mitochondrial matrix. NH 4 + is source of NH 3.NH 4 + is source of NH 3. The hydrolysis of two ATP make this reaction essentially irreversible.The hydrolysis of two ATP make this reaction essentially irreversible. N-acetyl glutamate is an allosteric activator. (see S08L05)N-acetyl glutamate is an allosteric activator. (see S08L05) 4 (p. 645)

6. 1. ARGININOSUCCINATE SYNTHASE 2. ARGININOSUCCINASE 3. ARGINASE 4. ORNITHINE TRANSCARBAMOYLASE 1. ARGININOSUCCINATE SYNTHASE 2. ARGININOSUCCINASE 3. ARGINASE 4. ORNITHINE TRANSCARBAMOYLASE 2 ~ P used 5

7. Connection to Krebs Cycle Fumarate is oxidized to oxaloacetate by Krebs cycle enzymes, producing NADH.Fumarate is oxidized to oxaloacetate by Krebs cycle enzymes, producing NADH. Oxaloacetate accepts an amino group instead of being condensed with acetyl CoA.Oxaloacetate accepts an amino group instead of being condensed with acetyl CoA. Fumarate is oxidized to oxaloacetate by Krebs cycle enzymes, producing NADH.Fumarate is oxidized to oxaloacetate by Krebs cycle enzymes, producing NADH. Oxaloacetate accepts an amino group instead of being condensed with acetyl CoA.Oxaloacetate accepts an amino group instead of being condensed with acetyl CoA. 6

8. Amino Acids to Urea * Glutamate Dehydrogenase is the control site: ADP (+), GDP (+), ATP (-), GTP (-) and NADH (-). * Glutamate Dehydrogenase is the control site: ADP (+), GDP (+), ATP (-), GTP (-) and NADH (-). Control at other sites by glucagon (+), cortisol (+), insulin (-), growth hormone (-). * Glutamate Dehydrogenase is the control site: ADP (+), GDP (+), ATP (-), GTP (-) and NADH (-). * Glutamate Dehydrogenase is the control site: ADP (+), GDP (+), ATP (-), GTP (-) and NADH (-). Control at other sites by glucagon (+), cortisol (+), insulin (-), growth hormone (-). 7

9. Summary of Reactions and Energetics - 1 H 2 0 + aa + NAD +   -keto acid + NH 4 + + NADH + H + and H 2 0 + fumarate + aa + NAD +  aspartate +  -keto acid + NADH + H + then aspartate + NH 4 + + HCO 3 - + 3 ATP  urea + fumarate + 2 H 2 0 + 2 ADP + AMP + 4 Pi + H + Four high energy phosphate bond equivalents are used for these reactions (- 4 ~P). Two NADH are produced. H 2 0 + aa + NAD +   -keto acid + NH 4 + + NADH + H + and H 2 0 + fumarate + aa + NAD +  aspartate +  -keto acid + NADH + H + then aspartate + NH 4 + + HCO 3 - + 3 ATP  urea + fumarate + 2 H 2 0 + 2 ADP + AMP + 4 Pi + H + Four high energy phosphate bond equivalents are used for these reactions (- 4 ~P). Two NADH are produced. 8

10. Summary of Reactions and Energetics - 2 Now consider NADH oxidation: 2 H + + 2 NADH + O 2  2 NAD + + 2 H 2 0 (+5 ~P) The net reaction is then 2 aa + HCO 3 - + O 2  2  -keto acid + urea + H + + 2 H 2 0 (+1~P) Now consider NADH oxidation: 2 H + + 2 NADH + O 2  2 NAD + + 2 H 2 0 (+5 ~P) The net reaction is then 2 aa + HCO 3 - + O 2  2  -keto acid + urea + H + + 2 H 2 0 (+1~P) 9

11. Hyperammonemia Normal blood [NH 4 + ] is 10-40  M.Normal blood [NH 4 + ] is 10-40  M. Deficiencies of carbamoyl phosphate synthetase or of any enzyme in the urea cycle cause high [NH 4 + ].Deficiencies of carbamoyl phosphate synthetase or of any enzyme in the urea cycle cause high [NH 4 + ]. Affects CNS and can lead to irreversible brain damage.Affects CNS and can lead to irreversible brain damage. Treatment strategies depend on which enzyme is deficient.Treatment strategies depend on which enzyme is deficient. Normal blood [NH 4 + ] is 10-40  M.Normal blood [NH 4 + ] is 10-40  M. Deficiencies of carbamoyl phosphate synthetase or of any enzyme in the urea cycle cause high [NH 4 + ].Deficiencies of carbamoyl phosphate synthetase or of any enzyme in the urea cycle cause high [NH 4 + ]. Affects CNS and can lead to irreversible brain damage.Affects CNS and can lead to irreversible brain damage. Treatment strategies depend on which enzyme is deficient.Treatment strategies depend on which enzyme is deficient. 10

12. Argininosuccinase Deficiency Low dietary protein reduces need for urea cycle.Low dietary protein reduces need for urea cycle. High dietary arginine provides a path for carbamoyl phosphate and aspartate nitrogens to produce argininosuccinate, which is excreted.High dietary arginine provides a path for carbamoyl phosphate and aspartate nitrogens to produce argininosuccinate, which is excreted. 11

13. Carbamoyl Phosphate Synthetase Deficiency Hippurate and phenylacetylglutamine are excreted.Hippurate and phenylacetylglutamine are excreted. Amino groups to glycine and glutamine by transamination.Amino groups to glycine and glutamine by transamination. Hippurate and phenylacetylglutamine are excreted.Hippurate and phenylacetylglutamine are excreted. Amino groups to glycine and glutamine by transamination.Amino groups to glycine and glutamine by transamination. 12 Fig. 23.20

14. Ketogenic and Glucogenic Amino Acids After removal of the amino group, the keto acids are used to make Krebs cycle intermediates, pyruvate, acetyl CoA and acetoacetyl CoA.After removal of the amino group, the keto acids are used to make Krebs cycle intermediates, pyruvate, acetyl CoA and acetoacetyl CoA. 13 Fig. 23.21

15. Nitrogen for Oral Bacteria Urea is a major source of nitrogen for oral bacteria.Urea is a major source of nitrogen for oral bacteria. It diffuses through most membranes and is in saliva.It diffuses through most membranes and is in saliva. Bacterial urease produces NH 4 +.Bacterial urease produces NH 4 +. Glutamate dehydrogenase incorporates NH 4 + into  -keto acids to obtain  amino acids for bacterial growth.Glutamate dehydrogenase incorporates NH 4 + into  -keto acids to obtain  amino acids for bacterial growth. Urea is a major source of nitrogen for oral bacteria.Urea is a major source of nitrogen for oral bacteria. It diffuses through most membranes and is in saliva.It diffuses through most membranes and is in saliva. Bacterial urease produces NH 4 +.Bacterial urease produces NH 4 +. Glutamate dehydrogenase incorporates NH 4 + into  -keto acids to obtain  amino acids for bacterial growth.Glutamate dehydrogenase incorporates NH 4 + into  -keto acids to obtain  amino acids for bacterial growth. 14

16. Nitrogen for Bacterial Amino Acid Synthesis When [NH 4 + ] is limiting, it does not bind glutamate dehydrogenase, and the lower two reactions are used.When [NH 4 + ] is limiting, it does not bind glutamate dehydrogenase, and the lower two reactions are used. 15

17. Engineered Oral Bacteria to Fight Caries? Streptococcus Salivarius urease activity affects oral microbial ecology.Streptococcus Salivarius urease activity affects oral microbial ecology. It produces NH 3, which in addition to promoting growth, neutralizes acids produces by other bacteria.It produces NH 3, which in addition to promoting growth, neutralizes acids produces by other bacteria. S. Salivarius urease gene was introduced into Streptococcus mutans GS5. It was expressed and during glucose metabolism reduced pH decrease and duration.S. Salivarius urease gene was introduced into Streptococcus mutans GS5. It was expressed and during glucose metabolism reduced pH decrease and duration. (Clancy & Burne,1997 FEMS Microbiol Lett 151:205) (Clancy & Burne,1997 FEMS Microbiol Lett 151:205) Streptococcus Salivarius urease activity affects oral microbial ecology.Streptococcus Salivarius urease activity affects oral microbial ecology. It produces NH 3, which in addition to promoting growth, neutralizes acids produces by other bacteria.It produces NH 3, which in addition to promoting growth, neutralizes acids produces by other bacteria. S. Salivarius urease gene was introduced into Streptococcus mutans GS5. It was expressed and during glucose metabolism reduced pH decrease and duration.S. Salivarius urease gene was introduced into Streptococcus mutans GS5. It was expressed and during glucose metabolism reduced pH decrease and duration. (Clancy & Burne,1997 FEMS Microbiol Lett 151:205) (Clancy & Burne,1997 FEMS Microbiol Lett 151:205) 16

18. Web links Nitrogen FixationNitrogen Fixation. A summary of the topic. Nitrogen Fixation Nitrogen CycleNitrogen Cycle. The biological big picture. Nitrogen Cycle Amino Acid MetabolismAmino Acid Metabolism. Reviews reactions. Amino Acid Metabolism Next topic: Porphyrins, heme, bile pigments Web links Nitrogen FixationNitrogen Fixation. A summary of the topic. Nitrogen Fixation Nitrogen CycleNitrogen Cycle. The biological big picture. Nitrogen Cycle Amino Acid MetabolismAmino Acid Metabolism. Reviews reactions. Amino Acid Metabolism Next topic: Porphyrins, heme, bile pigments