Lecture outline… Protein Metabolism II Urea Cycle 27th Mar 2012 Know the: 1.Urea Cycle: Reaction and Significance. 2.The Importance of the Cytosol and Mitochondria. 3.Enzyme Defect and Subsequent Clinical Diagnosis. 27th Mar 2012

Learning Objectives II.Urea Cycle A.Reactions of the urea cycle. 1.Synthesis of carbamoyl phosphate. 2. Production of arginine by urea cycle. 3. Cleavage of arginine to produce urea. B. Origin of ornithine. C. Regulation of the urea cycle. D. Function of the urea cycle during fasting. III. Disorders of the Urea Cycle. http://www.wiley.com/college/fob/quiz/quiz20/20-8.html 2

What are the Precursors of Urea What is Urea Urea is a diamide, chief nitrogenous waste product. What are the Precursors of Urea Ammonia +CO2 + Aspartate are the precursors of Urea. Other nitrogenous waste products which includes uric acid & creatinine. 3

Summary of Amino Acid Metabolism Dietary Proteins Digestion (Stomach Intestine) Amino Acids in Blood Membrane Other N-Containing Compounds Proteins Amino Acids Nitrogen Carbon g-Aminobutyrate Dopamine Norepinephrine Epinephrine Serotonin H2N-C-NH2 Urea O CO2 + H2O Energy ATP 4

Fate of Amino Acid Nitrogen: Urea Cycle Amino Acids Nitrogen Carbon Storage CO2+H2O NH4+ Energy ATP +CO2 COO- H C- - - NH2 -C- Urea O - NH2 NH2 Urea Cycle Aspartate What are the source of amino groups and carbonyl group for the biosynthesis of Urea?. 5

Most mammals convert amino-acid nitrogen to urea for excretion Excretion of Nitogenous Waste Products The carbon chains are broken down to molecules that feed into the TCA cycle. Amino acids Most mammals convert amino-acid nitrogen to urea for excretion NH4+ Some animals excrete NH4+ or uric acid. Fish & other aquatic vertebrates Most terrestrial vertebrates Birds & reptiles O HN N H H2N-C-NH2 Urea O NH4+ Ammonium ion Uric acid 6

An Overview of Urea Cycle What Are The Sites & The Organ In Which Biosynthesis Of Urea Occurs? Urea biosynthesis occurs partly in the cytosol and partly in the mitochondria of the liver. Net reaction: NH3 + CO2 + 2 ATP (HCO3) Carbamoyl-Phosphate + 2 ADP + Pi

phosphate synthase I (CPS-I) Transcarbamoylase (OTC) The Urea Cycle in the Liver Mitochondrial Matrix NH4+ + HCO3- + 2 ATP Carbamoyl phosphate synthase I (CPS-I) 1 Carbamoyl phosphate Ornithine Transcarbamoylase (OTC) 2 Ornithine Citrulline Hepatocyte Citrulline Aspartate Ornithine Argininosuccinate Synthetase ATP 3 AMP+ Pi Argininosuccinate 5 Argininosuccinate Lyase 4 Fumerate Arginase Arginine Cytoplasm Urea

What is Normal Value of Urea Is there any Specific functions of Urea Serum urea concentration is 15-40 mg/dL. Accumulation of urea more than normal value in blood is referred as’”Uremia”. Urea Excretion: 20–30 gm / day. Is there any Specific functions of Urea Not yet identified, but probably may involve in ‘’’’’the maintenance of osmotic balance as a ‘’’’’crystalloid. 9

Osmolality of Plasma Osmolality is a measure of the solute particles present in the fluid medium. Osmolality of Plasma: 285-295 milliosmoles / Kg. Plasma Osmolality can be computed from the concentrations (mmol/l) of Na+, K+, urea, & glucose as follows. 2(Na+) + 2(K+) + Urea + Glucose The factor 2 is used for Na+ & K+ ions for the associated anion concentration. Plasma Osmolality will be altered in lipidaemias, ESRD, hyperproteinaemia, ketoacidosis, diabetes insipidus,chronic alcohol intoxication, diuretics & in chronic diarrhoea.

Is any Clinical Conditions Are related with Low Urea Level Impairment of liver functions and congenital deficiencies of urea cycle enzymes will impair the biosynthesis of urea, which ultimately leads to accumulation of ammonia in blood and CSF causes “azotemia”. 11

What Is Meant By Azotemia Accumulation of nitrogenous materials including ammonia due to impairment in urea biosynthesis due to, Urea cycle enzyme deficiencies, Gastrointestinal tract (GI) bleeding, Valproic acid therapy and Renal impairments ect.,. 12

What Are The Consequences of Uremia Uremia will leads to ammonia toxicity, hypercapnia & alkalosis which may leads to neurotoxicity depending on the severity. Impaired renal function leads to erythropoietin deficiency and hence accumulation of CO2. 13

What Are The Causes of Uremia Ureamia may occur as a result of impairment in ‘’’’’the renal functions, which occur as a result of nephrotic syndrome, heavy metal toxicities, ESRD and nitrogen load due to high protein diet. 14

Importance of Urea Cycle Removal of CO2. Removal of Ammonia. Recycling of TCA cycle intermediates. Recycling of amino acids & ketoacids. 15

Involvement of Peripheral Tissues And Liver in the Ammonia Metabolism α-KG = α-Ketoglutaratee GDH = Glutamate Dehydrogenase.

Molecualr Interconversions in Handling of Ammonia α-Ketoglutarate Aspartate Alanine NH4+ NADH+H+ ALT NADPH+H+ Glutamate Dehydrogenase AST NADPH+ Pyruvate H2O NAD Glutamate Oxaloacetate NH4+ ATP Glutamine Synthetase ADP+Pi Glutamine The major enzyme responsible for the interconversion of Glutamate into α-KG is Glutamate Dehydrogenase. ALT= Alanine aminotransferase AST= Aspartate aminotransferase.

Reactions of the Urea Cycle Synthesis of Carbomoyl Phosphate. Production of Arginine by the Urea Cycle. Cleavage of Arginine to Produce Urea. (3) 18

Urea is less toxic than ammonia. Most terrestrial land animals convert excess nitrogen to urea, prior to excreting it. Urea is less toxic than ammonia. The Urea Cycle occurs mainly in liver. The 2 nitrogen atoms of urea derived from NH3 and the amino nitrogen of aspartate.

Carbamoyl Phosphate Synthase is the committed step of the Urea Cycle, and is subject to regulation. The NH3 and HCO3- that will be part of urea are incorporated first into carbamoyl phosphate (CP).

Carbamoyl Phosphate Synthase-I has an absolute requirement for an allosteric activator N-acetylglutamate. This derivative of glutamate is synthesized from acetyl-CoA & glutamate when cellular [glutamate] is high, signaling an excess of free amino acids due to protein breakdown or dietary intake.

Formation & Degradation of N-Acetylglutamate (NAG) N-Acetyl glucoseamine (NAG) serves as an allosteric activator for the enzyme CPS-I for the biosynthesis of carbamoyl phosphate (CP) with the help of HCO3- and ATP. NAG, is an allosteric activator of CPS-I.

The Reactions of the Urea Cycle Mitochondria NAG:N-acetyl glutamate; (in the formation of urea, one amino group is derived from free NH4+ ion, while the other is from aspartate. Carbon is obtained from CO2. (*) mitochondrial enzymes, the rest of the enzymes are cytosolic). Cytosol * NAG * 23

Urea Role of Glutamate in Urea Production Glutamate Urea Cycle Amino acids α-Ketoglutarate Transamination Other reactions Glutamate α-Ketoacids Transamination Oxalaoacetate α-Ketoglutarate NH4+ Aspartate Urea Cycle Urea 24

Role of Glutamate in Amino Acid Synthesis α-Ketoglutarate PLP GDH Transamination Glutamate α-Ketoacid PLP Amino acids α-Ketoglutarate Glutamate transfers Nitrogen by means of transamination reactions to α–ketoacids to form AAs. The nitrogen is obtained by glutamate either from transamination of other AAs or from NH4+ by means of the glutamate dehydrogenase (GDH) reaction PLP= Pyridoxal phosphate 25

Oxaloacetate is converted to aspartate via transamination (e. g Oxaloacetate is converted to aspartate via transamination (e.g., from glutamate). Aspartate then reenters Urea Cycle, carrying an amino group derived from another amino acid.

Regulation of the Urea Cycle Liver has a vast capacity to convert AA nitrogen to urea ,thereby preventing toxic effects from ammonia. Urea cycle is regulated by substrate (S) availability; the higher the rate of NH3 production, the higher the rate of urea formation. Regulation by the S availability is a general characteristics of disposal pathways, such as the urea cycle, which remove toxic compounds from the body. This is a type of “feed-forward” regulation, in contrast to the “feed-back” regulation characteristic of pathways that produce functional endproducts. 27

Regulation of the Urea Cycle… The other type of regulation control the urea cycle: Allosteric activation of CPS-I by N-acetylglutamate (NAG) and induction/repression of the synthesis of urea cycle enzymes. NAG is formed specifically to activate CPS-I; it has no other known function in mammals. The synthesis of NAG from acetyl CoA and glutamate is stimulated by arginine. Thus, the arginine level increase within the liver, 2 important reactions are stimulated. The first is the synthesis of NAG, which will increase the rate at which CP is produced. The 2nd is to produce more ornithine (via arginase rxn), such that the cycle can operate more rapidly. 28

D. Functions of Urea Cycle During Fasting During fasting, the liver maintains blood glucose by gluconeogenesis by utilizing muscle protein AAs as a carbon source. As AA carbons are converted to glucose, the nitrogens are converted to urea, thus the urinary excretion of urea is high during fasting. As fasting progresses, the brain begins to use ketone bodies, sparing blood glucose. Less muscle protein is cleaved to provide AAs for gluconeogenesis,and decreased production of glucose from AAs for gluconeogenesis. Hence, Decreased production of glucose from AAs is accompanied by decreased production of urea. 29

D.Functions of Urea Cycle During Fasting… The major AA substrate for gluconeogenesis is Alanine, which is synthesized in peripheral tissues to act as a nitrogen carrier. Glucagon release, which is expected during fasting, stimulates alanine transport into the liver by activating the transcription of transport systems for alanine. Two molecules of alanine are required to generate one molecule of glucose. The nitrogen from 2 molecules of alanine is converted to one molecule of urea. 30

Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood. Total lack of any Urea Cycle enzyme is lethal. Elevated ammonia is toxic, especially to the brain. If not treated immediately after birth, severe mental retardation results.

Genetic Deficiency of Urea Synthesis May be caused by, Ornithine Transcarbamylase Deficiency (OTC) Citrullinemia. Arginase Deficiency Argininosuccinic Aciduria Carbamyl Phosphate Synthetase (CPS-I) Deficiency N-Acetyl Glutamate Synthetase Deficiency (NAGS)

Carbamoyl Phosphate Synthetase-I Ornithine Transcarbamylase (OTC) Genetic Deficiency of Urea Synthesis Carbamoyl Phosphate Synthetase-I (CPS-I) Deficiency Ornithine Transcarbamylase (OTC) ↑[NH4+]; hyperammonemia ↑Blood Glutamine BUN is decreased No increase in uracil or orotic acid ↑Uracil & orotic acid in blood & urine Cerbral edema Cerebral edema Lethargy, convulsions, coma, death

Metabolic Diseases of the Urea Cycle Disorders present in infants: Symptoms: Lethargy, swelling of the brain leads to mental retardation / brain damage. Diagnosis: Low blood urea nitrogen (BUN) levels -high levels of ammonia in the blood elevated circulating glutamine -other metabolites that accumulate depend on the specific enzyme defect. Most common form: Hyperammonemia Type II caused by Ornithine Transcarbamylase (OTC) Deficiency elevated Carbamoyl-Phosphate (CP) levels in this deficiency cause secondary problems in pyrimidine metabolism.

Treatment: Long term, dietary restriction. Low protein diet. Supplemented with Arginine. Short term Dialysis. Administration of Nitrogen “scavengers”. e.g. Phenylacetate.

Treatment of Hyperammonemia with Phenylacetate: taking advantage of metabolism Phenylbutyrate

Glycine + Benzoic Acid Hippuric Acid Excreted in Urine

Review Q & As

Which biomolecule acts as a positive modulator for the first step of the urea cycle which is catalyzed by carbamoyl phosphate synthetase-I (CPS-I). A. Gultamate. B. Glutamine. C. N-Acetyl Glutamate (NAG). D. Aspartate. E. Ammonium ions (NH4+). Ans: C.

7. Which of the following is a common compound shared by The TCA cycle and the urea cycle? A. α-ketoglutarate. B. Succcinyl Coenzyme A (CoA). C. Oxaloacetate. D. Fumarate.* E. Arginine. Ans: D.

8. Which of the following enzymes requires adenosine triphosphate (ATP) to mediate its reactions? A. Argininosuccinate lyase. B. Argininosuccinate synthetase. C. Arginase. D. Glutaminase. E. Ornitine transcarbamoylase. Ans: B.

10. Two days after a full-term normal delivery, a neonate begins to hyperventilate, develops hypothermia and cerebral edema, and becomes comatose. Urinalysis reveals high levels of glutamine and orotic acid. The BUN is below normal. Which enzyme is most likely to be deficient in this child? A. Cytoplasmic glutaminase. B. Cytoplasmic carbamoyl phosphate synthetase. C. Cytoplasmic orotidylate decarboxylase. D. Mitochondrial carbamoyl phosphate synthetase. E. Mitochondrial ornitihine transcarbamoylase. Ans: E.

11. Transamination reactions are essential for ammonia assimilation 11. Transamination reactions are essential for ammonia assimilation. What cofactor is required to catalyze transamination reactions? A. pyridoxal phosphate (PLP). B. thiamin pyrophosphate. C. biotin. D. NAD+. E. NADPH. Ans: A.

12. Which of the following enzymes function in the biological assimilation of ammonia? A. Glutamate dehydrogenase. B. Glutamine synthetase. C. Glutamate synthase. D. All of the above.*** E. None of the above. Ans: D.

A 55-year-old man suffers from cirrhosis of the liver. Toxins such as ammonia are not properly metabolized by the liver and can now damage structures such as the brain. Which of the following amino acids covalently binds ammonia and transports and stores it in a nontoxic form? Aspartate. Glutamate. Serine. Cysteine. Histidine. Ans: B.

14. Which amino acid participates as a pathway intermediate in the urea cycle? Lysine. Arginine.*** Glutamine. Histidine. Tyrosine. Ans: B

Urea Cycle Must Know Key Points Urea cycle occurs partly in ……….. and partly in the ………. Ans: Mitochondria, Cytosol / Cytoplasm. 2. The rate-limiting step (committed step) in the urea cycle is catalyzed by the enzyme ………… Ans: Carbamoyl phosphate synthetase-I (CPS-I). 3. The biomolecule acts as a positive modulator in the first step of the urea cycle is…………… Ans: N-Acetyl glutamate (NAG). 4. ………….can be converted to arginine by a series of reactions, some of which requires urea cycle enzymes. Ans: Glutamate.

5. The enzymes are responsible for producing the direct donors of nitrogen into the pathway producing urea, include ………. And ……….. Ans: Aspartate aminotransferase and carbmoyl phosphate sythetase. 6. The accumulation of glutamine and orotic acid in the blood and an ultimate ammonia toxicity in the newborn is due to the deficiency of the urea cycle enzyme………. Ans: Mitochondrial ornitihine transcarbamoylase (OTC). 7. The common nitrogen acceptor for all reactions involving transaminase is …………… Ans: α-ketoglutarate. 8. The common nitrogen donor for all reactions involving Ans: Oxaloacetate (OAA).

9. The common compound shared by the TCA cycle and the urea cycle is ……………… Ans: Fumarate. 10. Hyperammonemia caused by a congenital defect of the urea cycle enzyme………..which is characterized by accumulation of excess amount of arginine in the blood. Ans: Arginase. 11. The urea cycle enzyme which requires adenosine triphosphate (ATP) to mediate its reactions is ?. Ans: Argininosuccinate synthetase. 12. The reaction catalyzed by an urea cycle enzyme ornithine aminotransferase which requires --------- as a coenzyme, which converts ornithine and α-ketoglutarate into glutamate. Ans: Pyridoxal phosphate (PLP).

Match the Urea Cycle Disorders With the Enzyme Defect No Disorder Answer Enzyme Defect 1 Hyperammonemia type I A. Argininosuccinate synthase 2 Hyperammonemia type II B. Ornithine transcarbomylase (OTC) 3 Citrullinemia C. Argininosuccinate lyase 4 Argininosuccinic Aciduria D. Arginase 5 Hyperargininemia E. Carbamoyl-P synthase-I (CPS) Answer: 1E; 2-B; 3-A; 4-C; 5-D

Genetic Deficiencies in Some of the Urea Cycle Enzymes can be Treated Pharmacologically by eliminating the amino acids such as glycine and glutamine by administering, A. Aspartic acid. B. Benzoic acid and phenylacetate. C. GABA. D. Pyridoxal phosphate. E. Methionine. Ans: B. The amide products of these reactions (hippurate and phenylacetylglutamine) are excreted in the urine. Synthesizing the Gly or Gln removes ammonia.

The Reaction Catalyzed by Glutamate Dehydrogenase which reversibly converts glutamate to α-ketoglutarate require the cofactor, ATP NAD NAD(P)+ /NAD(P)H. Biotin Pyridoxal phosphate. Ans: C. The cofactors require by the glutamate dehydrogenase is NAD(P)+ /NAD(P)H.