Transamination and urea cycle of protein metabolism.pptx

it describes the transamination of protein metabolism and also the urea cycle

1. Session objectives Students are expected to know what is amino acids Essential and non essentials amino acids Amino acid metabolism(Transamination, deamination's) How ammonia transported in the circulations Urea cycle and its regulation Urea cycle defect and ammonia toxicity

2. Amino acids Amino acids are organic acid having carboxylic acid (COOH) and amino(NH 2 ) functional group with side chain(R) different for all Unlike fats and carbohydrate amino acids are not stored in the body Therefore, amino acids must be obtained from the diet , synthesized de novo , normal protein degradation. Any amino acids in excess of the biosynthetic needs of the cell are rapidly degraded

3. Amino acids More than 300 different amino acids have been described in nature However, only twenty (20) are commonly found as constituents of mammalian proteins Except for proline, each amino acid has a carboxyl group, a primary amino group, and a distinctive side chain (“R-group”) bonded to the α-carbon atom Amino acids have amino ( pKa ~9.0-10.5) and carboxyl groups ( pKa ~2.0-2.4) that can be titrated At physiologic pH the carboxyl group is deprotonated (–COO - ), and the amino group is protonated (–NH 3 + ).

4. Structure of Amino acids Amino acids differ in size, shape, electrical charge from each other

5. Functions of amino acids  To form proteins Stabilize 3-D dimension structure of proteins by forming H and disulfide bonds Amino acid facilitate catalytic activity Precursors for Gluconeogenesis, C skeleton and N of amino acids used for the synthesis of purine and pyrimidine bases Histidine can be converted to histamine

6. Nutritional classification of amino acid Essentials • Arginine (Semi) • Histidine (semi) • Isoleucine • Leucine • Lysine • Methionine • Phenylalanine • Threonine • Tryptophan • Valine Non-essentials Alanine (from pyruvic acid) Asparagine (from aspartic acid) Aspartic Acid (from oxaloacetic acid) Cysteine Glutamic Acid (from oxoglutaric acid) Glutamine (from glutamic acid) Glycine (from serine and threonine) Proline (from glutamic acid) Serine (from glucose) Tyrosine (from phenylalanine)

7. Metabolic classifications

8. Metabolism of amino acid All amino acids are comprised of an amino group and a carbon skeleton . A.As are metabolized in two steps and make it more complex than CHO and FA metabolism The amino group can be used to synthesize new biomolecules , or it can be excreted The carbon skeleton will be used as energy source for the cells. Most amino acids are metabolized in the liver , but branched-chain amino acids leucine, isoleucine, and valine are exclusively metabolized in muscle.

9. Transamination The first step in the catabolism of most amino acids is the transfer of their α-amino group to α- ketoacid The products are an α-keto acid (derived from the original amino acid) and another amino acid. α-Ketoglutarate plays a pivotal role in amino acid metabolism by accepting the amino groups from other amino acids, thus becoming glutamate . Glutamate produced by transamination can be oxidatively deaminated or used as an amino group donor in the synthesis of nonessential amino acids.

10. Transamination Aminotransferase reaction is readily reversible and found in most cells, but are abundant in the liver. Aminotransferases enzymes are named after amino group donor amino acids. Because the acceptor of the amino group is almost always α- ketoglutarate . The pyridoxal phosphate (PLP) cofactor accepts and donates amino groups in chemical reactions.

11. Transamination reaction Transamination + deamination = trans-deamination

12. AST and ALT are the commonest aminotransferase The two most important aminotransferase reactions are catalyzed by alanine aminotransferase (ALT) and aspartate aminotransferase AST

13. Mechanism of action of aminotransferase All aminotransferases require the coenzyme pyridoxal phosphate (a derivative of vitamin B6. It covalently linked to the ε-amino group of a specific lysine residue at the active site of the enzyme. It then transferring the amino group of an amino acid to the pyridoxal part of the coenzyme to generate pyridoxamine phosphate. The pyridoxamine form of the coenzyme then reacts with an α-keto acid to form an amino acid, at the same time regenerating the original aldehyde form of the coenzyme

14. Mechanism of action of aminotransferase

15. B. Trans-deamination Transamination takes place in the cytoplasm of all the cells of the body Then after the amino group is transported to liver as glutamic acid Glutamate then oxidatively deaminated in the mitochondria of hepatocytes . Thus, the two components of the reaction are physically far away, but physiologically they are coupled. Hence, the term trans-deamination reaction is the final reaction which removes the amino group of all amino acids

16. Trans-deamination The glutamate dehydrogenase catalyze oxidative deamination. This reaction is unusual in that it can use both NAD+ or NADP+ as cofactors.

17. Glutamate dehydrogenase

18. Some amino acids don’t undergo aminotransferase at all; they just have their amino groups removed directly. This includes threonine and serine , which are nonoxidative deaminated by threonine dehydratase and serine dehydratase, respectively.

19. Biological Significance of Transamination 1. Removal of ammonia In this first step, ammonia is removed, and the carbon skeleton of the amino acid enters into catabolic pathway. 2. Synthesis of nonessential amino acids By means of transamination, all nonessential amino acids can be synthesized by the body from keto acids available from other sources. Pyruvate can be transaminated to synthesize alanine Oxaloacetate produces aspartic acid . Alpha keto glutarate is transaminated to form glutamic acid .

20. Cont.…. 3. Interconversion of amino acids/amino acids equalizations If amino acid no.1 is high and no.2 is low; the amino group from no.1 may be transferred to a keto acid to give amino acid no. 2 to equalize the quantity of both. This is called equalization of quantities of nonessential amino acids.

21. Minor Pathways of Deamination 1. D-Amino acid oxidase : D-Amino acids are found in plants and in the cell walls of microorganisms, but are not used in the synthesis of mammalian proteins. D-Amino acids are, however, present in the diet, and are efficiently metabolized by the kidney and liver. D-Amino acid oxidase is an FAD-dependent peroxisomal enzyme that catalyzes the oxidative deamination of these amino acid isomers. The resulting α-keto acids can enter the general pathways of amino acid metabolism, and be re- aminated to L-isomers, or catabolized for energy.

22. Diagnostic value of plasma aminotransferases Aminotransferases are normally intracellular enzymes Low levels found in the plasma represent the release during normal cell turnover. Normal value of ALT 7 to 55 U/L and AST 8 to 48 U/L Aspartate aminotransferase (AST)  is found in a variety of tissues, including the liver, brain,  pancreas , heart,  kidneys , lungs, and skeletal muscles. If any of these tissues are damaged, AST will be released into the bloodstream. While high AST levels mean there may be tissue injury, it doesn't always relate to the liver

23. Diagnostic value of plasma aminotransferases By contrast,  alanine aminotransferase (ALT)  is found mainly in the liver. Plasma AST and ALT are elevated in nearly all liver diseases, but are particularly high in conditions that cause extensive cell necrosis, such as severe viral hepatitis, toxic injury, and prolonged circulatory collapse. ALT is more specific than AST for liver disease, but the AST is more sensitive because the liver contains larger amounts of AST

24. Specificity vs sensitivity Sensitivity (true positive rate) is probability of a positive test It refers to a test's ability to designate an individual with disease as positive. The specificity(true negative) of a test is its ability to designate an individual who does not have a disease as negative . the probability of a negative test

25. Major fate of Ammonia ( Urea Cycle)

26. Fates of the amino group(NH3+) The amino group (now in ammonium form) has multiple fates: It can be excreted through the urea cycle It can be used to synthesize new biomolecules Biosynthesis of amino acids Biosynthesis of nucleotides Biosynthesis of biological amines Histamine Dopamine Melatonin

27. How is ammonium transported from peripheral tissues to the liver for excretion? It can’t be through the blood, as ammonium is toxic and acidic. Instead, glutamine synthetase combines ammonium and glutamate to produce glutamine. Glutamine is not toxic and so can be transported in the blood. Once in the liver the glutaminase reaction, which only occurs in liver mitochondria, converts glutamine back to glutamate and ammonium. The ammonium can then enter the urea cycle.

28. How intracellular NH 3 + is transported to liver 1. Glutamate-glutamine system This found in most tissues, uses glutamine synthetase to combine ammonia with glutamate to form glutamine—a nontoxic transport form of ammonia Then glutamine is transported in the blood to the liver where it is cleaved by glutaminase to produce glutamate and free ammonia. The ammonia thus generated is immediately detoxified into urea. The concentration of glutamic acid in blood is 10 times more than other amino acids. Glutamine is the transport forms of ammonia from brain and intestine to liver.

29. Glutamate-glutamine system

30. Glutamate –glutamine cycle

31. Cont.…. 2. Glucose-alanine system The second transport mechanism, used primarily by muscle, involves transamination of pyruvate (the end product of aerobic glycolysis) to form alanine Alanine is transported by the blood to the liver and converted back to pyruvate by transamination In the liver, the pathway of gluconeogenesis can use the pyruvate to synthesize glucose. Then glucose enter the blood and be used by muscle—a pathway called the glucose-alanine cycle .

32. Glucose- Alanine system

33. Sources of ammonia 1. Amino acids : Amino acids are quantitatively the most important source of ammonia. 2. From glutamine : The kidneys form ammonia from glutamine by the actions of renal glutaminase and glutamate dehydrogenase. Most of this ammonia is excreted into the urine as NH 4 + , for acid- base balance 3. From bacterial action in the intestine: Ammonia is formed from urea by the action of bacterial urease in the lumen of the intestine. 4. From amines : Amines obtained from the diet, and monoamines that serve as hormones or neurotransmitters, give rise to ammonia by the action of amine oxidase 5. From purines and pyrimidines :

34. Urea cycle Ammonia (NH3) is a colorless irritant gas readily soluble in water to generate ammonium (NH4+) ions. The blood ammonia level in a healthy adult is in a range of 5 to 50 micrograms/ dL . As a result of the highly toxic nature of ammonia, it is quickly metabolized into urea in the liver by the urea cycle and excreted by the kidneys. Ammonia toxicity occurs when the ammonia content in the blood supersedes the liver’s capacity to eliminate it; This could be a result of either overproduction such as in congenital hyperammonemia or under-elimination such as in liver cirrhosis.

35. Where do urea cycle reaction occur Urea is principally the end product of protein metabolism and take place in exclusively in liver The first two reactions leading to the synthesis of urea occur in the liver mitochondria Whereas the remaining three cycle enzymes are located in the cytosol The two nitrogen atoms of urea are derived from two different sources, one from ammonia and the other directly from the alpha amino group of aspartic acid .

36. Steps of urea cycle Formation of carbamoyl phosphate (in mitochondria) The first ammonia is added from glutamate by mitochondrial glutamate dehydrogenase One molecule of ammonia condenses with CO 2 in the presence of two molecules of ATP to form carbamoyl phosphate. CO 2 is the source of carbon, NH3 is source of N and ATP as source of Phosphate for carbamoyl phosphate The reaction is catalyzed by the mitochondrial enzyme carbamoyl phosphate synthetase-I (CPS-I). Carbamoyl phosphate synthetase I requires N- acetylglutamate as a positive allosteric activator

37. 1 st step of urea synthesis N-acetyl-glutamate synthesis Carbamoyl phosphate

38. 2. Formation of Citrulline (in mitochondria) Ornithine and citrulline are basic amino acids that participate in the urea cycle. They are not incorporated into cellular proteins, because there are no codons for these amino acids Ornithine is regenerated with each turn of the urea cycle The carbamoyl group of carbamoyl phosphate is transferred to the NH 2 group of ornithine by ornithine transcarbamoylase (OTC) The end product of this reaction is citrulline , that transported to the cytosol for next reaction

39. 3 . Synthesis of arginosuccinate (in cytosol) Citrulline condenses with aspartate to form arginosuccinate by Arginosuccinate synthetase enzyme. The α-amino group of aspartate provides the second nitrogen that is ultimately incorporated into urea. The formation of arginosuccinate is driven by the cleavage of ATP to adenosine monophosphate (AMP) and pyrophosphate. This 3rd reaction required ATP and two high energy phosphate bonds. The PPi is an inhibitor of this step and urea cycle use 4ATP per cycle

40. 4. Cleavage of arginosuccinate Arginosuccinate is cleaved by arginosuccinate lyase to arginine and fumarate The fumarate formed may be funneled into TCA cycle to be converted to malate and then to oxaloacetate to be transaminated to aspartate As a result urea cycle is linked to TCA-cycle through fumarate

41. 5. Cleavage of arginine to ornithine and urea (cytosol) Arginase cleaves arginine to ornithine and urea, and occurs almost exclusively in the liver. The ornithine returns to the mitochondria to react with another molecule of carbamoyl phosphate so that the cycle will proceed. Thus, ornithine may be considered as a catalyst which enters the reaction and is regenerated.

42. Urea cycle

43. Energetics of Urea Cycle The overall reaction may be summarized as: During these reactions, 2 ATPs are used in the 1 st reaction. Another ATP is converted to AMP + PPi in the 3 rd step, which is equivalent to 2 ATPs. The urea cycle consumes 4 high energy phosphate bonds. However, fumarate formed in the 4th step may be converted to malate. Malate when oxidized to oxaloacetate produces 1 NADH equivalent to 2.5 ATP. So net energy expenditure is only 1.5 high energy phosphates .

44. Fate of urea 1. Urea diffuses from the liver, and is transported in the blood to the kidneys, where it is filtered and excreted in the urine. 2. A portion of the urea diffuses from the blood into the intestine, and is cleaved to CO 2 and NH 3 by bacterial urease. This ammonia is partly lost in the feces, and is partly reabsorbed into the blood. In patients with kidney failure, plasma urea levels are elevated, promoting a greater transfer of urea from blood into the gut.

45. Fates of urea The intestinal action of urease on this urea becomes a clinically important source of ammonia, contributing to the hyperammonemia often seen in these patients. Oral administration of neomycin reduces the number of intestinal bacteria responsible for this NH 3 production.

46. Regulation of the Urea Cycle Nutritional state (Increase protein ingestion and starvation) During starvation, the rate of protein catabolism increased and increase the activity of urea cycle enzymes The intrahepatic concentration of N- acetylglutamate and glutamate increases during ingestion of a protein-rich meal. This leads to an increased rate of urea synthesis. Enzyme Carbamoyl phosphate synthetase - I, the rate-limiting step in the urea cycle is is activated by N- Acetylglutamate . N- Acetylglutamate is activated by arginine

47. Disorders of Urea Cycle The normal levels of serum ammonia are normally low (5–50 µmol/L). However, when liver function is compromised, due either to genetic defects of the urea cycle, or liver disease, blood levels can rise above 1,000 µ mol /L Ammonia is toxic to the nervous system, and its concentration in the body must be carefully controlled. Under normal conditions, free ammonia is rapidly fixed into either α-ketoglutarate (by glutamate dehydrogenase, to form glutamate) or glutamate (by glutamine synthetase , to form glutamine).

48. Disorder of urea cycle When the block is in one of the earlier steps, the condition is more severe, since ammonia itself accumulates. Deficiencies of later enzymes result in the accumulation of other intermediates which are less toxic and hence symptoms are less. All of the defects are autosomal recessive, except for ornithine transcarbomylase deficiency, which is X-linked recessive and the most common The most common urea-cycle defect is OTC deficiency, which is an X-linked disorder.

49. Hepatic encephalopathy Hepatic encephalopathy is a syndrome of neuropsychiatric manifestations in patients with liver disease, and one of the mechanisms of its development is hypothesized to be hyperammonemia . 10% of the patients with hepatic encephalopathy, however, do not have raised levels of ammonia Neurotoxicity from hyperammonemiama be due to Increased synthesis of glutamate from a- KG Increased synthesis of glutamine ( increased osmotic effect) Aalpha -ketoglutarate is depleted from CNS Inhibition of TCA cycle and production of less ATP

50. Urea cycle disorders

51. Treatment of urea cycle disorder Low-protein diets are essential to reduce the potential for excessive amino acid degradation. If the enzyme defect in the urea cycle comes after the synthesis of argininosuccinate, massive arginine supplementation has proved beneficial. Once argininosuccinate has been synthesized, the two nitrogen molecules destined for excretion have been incorporated Thus, ingesting large levels of arginine leads to ornithine production by the arginase reaction, and nitrogen excretion via argininosuccinate in the urine can be enhanced

52. cont... Arginine therapy will not work for enzyme defects that exist in steps before the synthesis of argininosuccinate. For these disorders, drugs are used that form conjugates with amino acids The conjugated amino acids are excreted, and the body then has to use its nitrogen to resynthesize the excreted amino acid. The two compounds most frequently used are benzoic acid and phenylbutyrate

53. cont.. Benzoic acid reacts with glycine to form hippuric acid, which is excreted. As glycine is synthesized from serine, the body now uses nitrogen to synthesize serine, so more glycine can be produced. Phenylacetate forms a conjugate with glutamine , which is excreted. This conjugate removes two nitrogen per molecule and requires The body resynthesize glutamine from akg , thereby using another two nitrogen molecules.

54. Treatment of Urea cycle defect

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