Metabolism of Nitrogenous Compound

1. Metabolism of Nitrogenous Compound Mpenda F.N 1

2. Introduction • This topic describes the amino acids that are important in human nutrition. • It covers the digestion and absorbtion of proteins in the gut. • The amino acid degradation pathways in the tissues, and the urea cycle. • The amino acid synthesis pathways • There are separate topic dealing with nucleotide and 'one carbon' metabolism and with porphyrin on the integration of metabolism. 2

3. Introduction… • Human proteins have very different lifetimes. • Total body protein is about 11kg, but about 25% of this is collagen, which is metabolically inert. • A typical muscle protein might survive for three weeks, but many liver enzymes turn over in a couple of days. • Some regulatory enzymes have half-lives measured in hours or minutes. • The majority of the amino acids released during protein degradation are promptly re-incorporated into fresh proteins. 3

4. Introduction… • Net protein synthesis accounts for less than one third of the dietary amino acid intake, even in rapidly growing children consuming a minimal diet. • Most of the ingested protein is ultimately oxidised to provide energy, and the surplus nitrogen is excreted, a little as ammonia but mostly as urea. 4

5. Protein degradation • Soluble intracellular proteins are tagged for destruction by attaching ubiquitin, a low molecular weight protein marker. • They are then degraded in proteasomes to short peptides. • A very few of these are displayed on the cell surface by the MHC [major histocompatibility] complex as part of the immune system, • But most of them are further metabolised to free amino acids. • Some proteins are degraded by an alternative system within the lysosomes. 5

6. Protein degradation • Dietary proteins are initially denatured by the stomach acid, in conjunction with limited proteolysis by pepsin. • In young mammals gastric rennin partially hydrolyses and precipitates milk casein and increases gastric residence time. • Gastric acid also kills most ingested bacteria, rendering the upper part of the gut almost sterile. 6

7. Protein digestion is largely completed in the small intestine at a slightly alkaline pH. • The pancreatic proteases trypsin, chymotrypsin and elastase divide the proteins into short peptides. • These are attacked from both ends by aminopeptidase and carboxypeptidase, and the fragments are finished off by dipeptidases secreted from the gut wall. Protein degradation 7

8. Amino acid catabolism • In animals, amino acids undergo oxidative degradation in three different metabolic circumstances: • During the normal synthesis and degradation of cellular proteins. • When a diet is rich in protein and the ingested amino acids exceed the body’s needs for protein synthesis. • During starvation or in uncontrolled diabetes mellitus, when carbohydrates are either unavailable or not properly utilized, cellular proteins are used as fuel. 8

9. Amino acid catabolism • The liver is the principal site of amino acid metabolism. • Other tissues, such as the kidney, the small intestine, muscles, and adipose tissue, take part. • The first step in the breakdown of amino acids is the separation of the amino group from the carbon skeleton, usually by a transamination reaction. • The carbon skeletons resulting from the deaminated amino acids are used to form either glucose or fats, or they are converted to a metabolic intermediate that can be oxidized by the citric acid cycle. 9

10. Amino acid catabolism • Under all these metabolic conditions, amino acids lose their amino groups to form α-keto acids, the “carbon skeletons” of amino acids. • The α-keto acids undergo oxidation to CO2 and H2O • More importantly provide three- and four-carbon units that can be converted by gluconeogenesis into glucose, the fuel for brain, skeletal muscle, and other tissues 10

11. Amino acid catabolism • Therefore, the processes of amino acid degradation converge on the central catabolic pathways. • The carbon skeletons of most amino acids finding their way to the citric acid cycle. • In some cases the reaction pathways of amino acid breakdown closely parallel steps in the catabolism of fatty acids. 11

12. Amino acid catabolism • The pathways for amino acid degradation include a key step in which the α-amino group is separated from the carbon skeleton and shunted into the pathways of amino group metabolism • We will deal first with amino group metabolism and nitrogen excretion. • We will then deal with the fate of the carbon skeletons derived from the amino acids; along the way we see how the pathways are interconnected. 12

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14. Metabolic Fates of Amino Groups • Excess ammonia generated in other (extrahepatic) tissues travels to the liver for conversion to the excretory form. • Glutamate and glutamine play especially critical roles in nitrogen metabolism, acting as a kind of general collection point for amino groups. • In the cytosol of hepatocytes, amino groups from most amino acids are transferred to α-ketoglutarate to form glutamate, which enters mitochondria and gives up its amino group to form NH4. 14

15. Excess ammonia generated in most other tissues is converted to the amide nitrogen of glutamine, which passes to the liver, then into liver mitochondria. • Glutamine or glutamate or both are present in higher concentrations than other amino acids in most tissues. • In skeletal muscle, excess amino groups are generally transferred to pyruvate to form alanine, another important molecule in the transport of amino groups to the liver. Metabolic Fates of Amino Groups 15

16. Central role of glutamate • Glutamate also occupies a special position in amino acid breakdown • Most of the nitrogen from dietary protein is ultimately excreted from the body via the glutamate pool. • Glutamate is special because it is chemically related to 2-oxoglutarate (= α-keto glutatarate) which is a key intermediate in the citric acid (Krebs) cycle. • Glutamate can be reversibly converted into 2-oxoglutarate by transaminases or by glutamate dehydrogenase. • In addition, glutamate can be reversibly converted into glutamine, an important nitrogen carrier, and the most common free amino acid in human blood plasma. 16

17. Central role of glutamate 17

18. Central role of glutamate 18

19. Because of the participation of 2-oxoglutarate in numerous transaminations, glutamate is a prominent intermediate in nitrogen elimination as well as in anabolic pathways. • Glutamate, formed in the course of nitrogen elimination, is either oxidatively deaminated by liver glutamate dehydrogenase forming ammonia, or converted to glutamine by glutamine synthetase and transported to kidney tubule cells. • There the glutamine is sequentially deamidated by glutaminase and deaminated by kidney glutamate dehydrogenase. Central role of glutamate 19

20. Central role of glutamate 20

21. The ammonia produced in the latter two reactions is excreted as NH4+ in the urine, where it helps maintain urine pH in the normal range of pH 4 to pH 8. • The extensive production of ammonia by peripheral tissue or hepatic glutamate dehydrogenase is not feasible because of the highly toxic effects of circulating ammonia. • Normal serum ammonium concentrations are in the range of 20–40μM, and an increase in circulating ammonia to about 400μM causes alkalosis and neurotoxicity. Central role of glutamate 21

22. Urea biosynthesis occurs in four stages: • Transamination • oxidative deamination of glutamate • ammonia transport • reactions of the urea cycle Biosynthesis of urea 22

23. Transamination reactions • The first step in the catabolism of most L-amino acids, once they have reached the liver, is removal of the α-amino groups, promoted by enzymes called aminotransferases or transaminases. • In these transamination reactions, the -amino group is transferred to the -carbon atom of alpha-ketoglutarate, leaving behind the corresponding -keto acid analog of the amino acid • The effect of transamination reactions is to collect the amino groups from many different amino acids in the form of L-glutamate 23

24. Transamination reactions • Transamination is an exchange of functional groups between any amino acid (except lysine, proline, and threonine) and an α-keto acid. • The amino group is usually transferred to the keto carbon atom of pyruvate, oxaloacetate, or α-ketoglutarate, converting the α-keto acid to alanine, aspartate, or glutamate, respectively. • Transamination reactions are catalyzed by specific transaminases (also called aminotransferases), which require pyridoxal phosphate as a coenzyme. 24

25. Transamination reactions 25

26. Transamination reactions Inmanyaminotransferase reactions, α -ketoglutarate is the amino group acceptor. All aminotransferases have pyridoxal phosphate (PLP) as cofactor. The reaction is readily reversible. 26

27. The role of Pyridoxal Phosphate in the Transfer of-Amino Groups to alpha-Ketoglutarate • All aminotransferases have the same prosthetic group and the same reaction mechanism. • The prosthetic group is pyridoxal phosphate (PLP), the coenzyme form of pyridoxine, or vitamin B6. • Also we saw pyridoxal phosphate in as a coenzyme in the glycogen phosphorylase reaction. • Its primary role in cells is in the metabolism of molecules with amino groups. 27

28. Pyridoxal phosphate functions as an intermediate carrier of amino groups at the active site of aminotransferases. • It undergoes reversible transformations between its aldehyde form, pyridoxal phosphate, which can accept an amino group, and its aminated form, pyridoxamine phosphate, which can donate its amino group to an α-keto acid. The role of Pyridoxal Phosphate in the Transfer ofα-Amino Groups to α-Ketoglutarate 28

29. L-GLUTAMATE DEHYDROGENASE OCCUPIES A CENTRAL POSITION IN NITROGEN METABOLISM • Transfer of amino nitrogen to α-ketoglutarate forms L-glutamate. • Release of this nitrogen as ammonia is then catalyzed by hepatic L-glutamate dehydrogenase • (GDH), which can use either NAD+ or NADP+ • Conversion of α-amino nitrogen to ammonia by the concerted action of glutamate aminotransferase and GDH is often termed “transdeamination” 29

30. L-GLUTAMATE DEHYDROGENASE OCCUPIES A CENTRAL POSITION IN NITROGEN METABOLISM The L-glutamate dehydrogenase reaction. NAD(P)+ means that either NAD+ or NADP+ can serve as co-substrate. The reaction is reversible but favors glutamate formation. 30

31. Glutamate dehydrogenase operates at an important intersection of carbon and nitrogen metabolism. • An allosteric enzyme with six identical subunits, its activity is influenced by a complicated array of allosteric modulators. • The best-studied of these are the positive modulator ADP and the negative modulator GTP • Mutations that alter the allosteric binding site for GTP or otherwise cause permanent activation of glutamate dehydrogenase lead to a human genetic disorder called hyperinsulinism-hyperammonemia L-GLUTAMATE DEHYDROGENASE OCCUPIES A CENTRAL POSITION IN NITROGEN METABOLISM 31

32. Serum aminotransferases such as aspartate aminotransferase, AST (also called serum glutamate-oxaloacetate transaminase, SGOT) and alanine transaminase, ALT (also called serum glutamate-pyruvate transaminase (SGPT) have been used as clinical markers of tissue damage, • with increasing serum levels indicating an increased extent of damage. • As indicated earlier, ALT has an important function in the delivery of skeletal muscle carbon and nitrogen (in the form of alanine) to the liver in a series of reactoins referred to as the glucose-alanine cycle L-GLUTAMATE DEHYDROGENASE OCCUPIES A CENTRAL POSITION IN NITROGEN METABOLISM 32

33. Glutamine Transports Ammonia in the Bloodstream • Ammonia is quite toxic to animal ,and the levels present in blood are regulated. • In many tissues, including the brain, some processes such as nucleotide degradation generate free ammonia. • In most animals much of the free ammonia is converted to a nontoxic compound before export from the extrahepatic tissues into the blood and transport to the liver or kidneys. • For this transport function, glutamate, critical to intracellular amino group metabolism, is supplanted by L-glutamine. • The free ammonia produced in tissues is combined with glutamate to yield glutamine by the action of glutamine synthetase. 33

34. Glutamine Transports Ammonia in the Bloodstream 34

35. The glutamine is sequentially deamidated by glutaminase and deaminated by kidney glutamate dehydrogenase. • The ammonia produced in the kidney reactions is excreted as NH4+ in the urine. • This helps maintain urine pH in the normal range of pH 4 to pH 8. • The extensive production of ammonia by peripheral tissue or hepatic glutamate dehydrogenase is not feasible because of the highly toxic effects of circulating ammonia. • Normal serum ammonium concentrations are in the range of 20–40μM, and an increase in circulating ammonia to about 400μM causes alkalosis and neurotoxicity. Glutamine Transports Ammonia in the Bloodstream 35

36. Glucose-alanine cycle • Alanine also plays a special role in transporting amino groups to the liver in a nontoxic form, via a pathway called the glucose-alanine cycle • In muscle and certain other tissues that degrade amino acids for fuel, amino groups are collected in the form of glutamate by transamination. • Glutamate can transfer its -amino group to pyruvate, a readily available product of muscle glycolysis, by the action of alanine aminotransferase. 36

37. The alanine so formed passes into the blood and travels to the liver. • In the cytosol of hepatocytes alanine aminotransferase transfers the amino group • from alanine to alpha-ketoglutarate, forming pyruvate and glutamate Glucose-alanine cycle 37

38. Glucose-alanine cycle 38

39. Urea cycle • About 80% of the excreted nitrogen is in the form of urea is produced exclusively in the liver, in a series of reactions that are distributed between the mitochondrial matrix and the cytosol. • The series of reactions that form urea is known as the Urea Cycle or the Krebs-Henseleit Cycle. • In the urea cycle, amino groups of urea are donated by carbamoyl phosphate and aspartate, while the carbon atom of urea is contributed by bicarbonate. 39

40. The essential features of the urea cycle reactions and their metabolic regulation are as follows: • Arginine from the diet or from protein breakdown is cleaved by the cytosolic enzyme arginase, generating urea and ornithine. • In subsequent reactions of the urea cycle a new urea residue is built on the ornithine, regenerating arginine and perpetuating the cycle. Urea cycle 40

41. Arginine 41

42. 42

43. The net reaction for urea synthesis shows consumption of 4 "high energy" phosphoanhydride bonds, contributed by ATP. • Two of these are used by for synthesis of carbamoyl phosphate from bicarbonate and ammonia. • The ammonia is itself ultimately derived from various amino acids by the combined action of transaminase enzymes and glutamate dehydrogenase. • Carbamoyl phosphate synthesis occurs in the mitochondrial matrix, and is catalyzed by carbamoyl phosphate synthetase I. Urea cycle 43

44. The carbamoyl phosphate produced is then consumed in the synthesis of citrulline from ornithine. • This reaction is catalyzed by ornithine carbamoyltransferase. • The citrulline is shuttled out of the mitochondrion and into the cytosol, where the rest of the urea cycle takes place. • Another amino acid-derived amino group is incorporated into the intermediate Aspartate is joined via its α-amino group to citrulline in the reaction catalyzed by argininosuccinate synthase to form argininosuccinate. Urea cycle 44

45. The next reaction is the elimination of fumarate from argininosuccinate, yielding arginine. • This step is catalyzed by argininosuccinase. • Finally, urea is produced by arginase, acting on arginine and water as substrates. • Urea is secreted into the bloodstream, from which it is ultimately eliminated by the kidneys for excretion. • The ornithine produced in this last step is shuttled into the mitochondrial matrix, completing the cycle Urea cycle 45

46. Regulation of the Urea Cycle • The urea cycle operates only to eliminate excess nitrogen. • On high-protein diets the carbon skeletons of the amino acids are oxidized for energy or stored as fat and glycogen, but the amino nitrogen must be excreted. • Enzymes of the urea cycle are controlled at the gene level. • long-term changes in the quantity of dietary protein, changes of 20-fold or greater in the concentration of cycle enzymes are observed. 46

47. When dietary proteins increase significantly, enzyme concentrations rise. • On return to a balanced diet, enzyme levels decline. • Under conditions of starvation, enzyme levels rise as proteins are degraded and amino acid carbon skeletons are used to provide energy, thus increasing the quantity of nitrogen that must be excreted. • Short-term regulation of the cycle occurs principally at CPS-I, which is inactive in the absence of its obligate activator N-acetylglutamate. • The steady-state concentration of N-acetylglutamate is set by the concentration of its components acetyl-CoA and glutamate and by arginine, which is a positive allosteric effector of N-acetylglutamate synthase. Regulation of the Urea Cycle 47

48. Read on molecular basis of ammonia intoxication 48

49. Krebs bicycle • Because the fumarate produced in the argininosuccinase reaction is also an intermediate of the citric acid cycle. • each cycle can operate independently and communication between them depends on the transport of key intermediates between the mitochondrion and cytosol. • The fumarate generated in cytosolic arginine synthesis can therefore be converted to malate in the cytosol, and these intermediates can be further metabolized in the cytosol or transported into mitochondria for use in the citric acid cycle 49

50. Urea Cycle Disorders (UCDs) • A complete lack of any one of the enzymes of the urea cycle will result in death shortly after birth. • However, deficiencies in each of the enzymes of the urea cycle, including N-acetylglutamate synthase, have been identified. • These disorders are referred to as urea cycle disorders or UCDs. • Take your time read on UCDs 50