Amino Acid Metabolism and Urea Cycle Notes

Dr. M. Bayramzadeh

Medicine

Biochemistry

1Oxidation-Reduction Reactions

 Oxidation: loss of electron or hydrogen (H) or the gain of oxygen (O2)

 Reduction: loss of oxygen or gain of hydrogen or electron

 Whenever one substance is oxidized, another substance is reduced

 Oxidized Substance: Lose Energy

 Reduced Substance: Gain Energy

 Coenzymes act as hydrogen (or electron) acceptors

 NAD/NADH NADP/NADPH

 FAD/FADH

23Digestion of proteins

 The dietary proteins are denatured during cooking to be more easily digested by

digestive enzymes.

 Proteolytic enzymes are secreted as inactive zymogens which are converted to

their active form in the intestinal lumen.

 This would prevent auto digestion of the secretory acini.

4Proteolytic enzymes include:

1. Endopeptidases: (Pepsin, Renin, Trypsin, Chymotrypsin, and Elastase).

 act on peptide bonds inside the protein molecule

 Convert the protein into smaller and smaller units.

2. Exopeptidases:

act at the peptide bond only at the end region of the chain.

a) Carboxypeptidase: act on peptide bond only at the carboxyl terminal end

b) Aminopeptidase: at the amino terminal end of the chain.

56Protein

Digestion

78A. Gastric digestion of proteins:

A. Stomach

a) Hydrochloric Acid

 It makes the pH optimum for pepsin & activates pepsin.

 The acid also denatures the proteins.

 But hydrochloric acid at body temperature could not break the peptide bonds.

 Thus, in the stomach, HCl lonely will not be able to digest proteins; it needs

enzymes.

9Pepsin:

 Endopeptidase.

 Pepsinogen is secreted by the chief cells of stomach.

 The HCL converts pepsinogen to pepsin.

 The optimum pH for activity of pepsin is around 2 (1.8-3.5).

10Rennin:

 Rennin or Chymosin

 Infants (active) and adults (absent).

 involved in the curdling of milk.

 Milk protein (casein) is converted to Paracasein by the action of rennin.

 The denatured protein is easily digested further by pepsin.

11B. Pancreatic digestion of proteins:

 The optimum pH (pH 8)

 Provided by the alkaline bile and pancreatic juice.

 The secretion of pancreatic juice is stimulated by

 the peptide hormones

 Cholecystokinin

 Pancreozymin.

 Pancreatic juice contains endopeptidases

 Trypsin

 Chymotrypsin

 Elastase

 Carboxypeptidase

12Pancreatic Enzymes

131) Trypsin:

 The activation of pepsin: two stages process

 Trypsinogen → Trypsin (by enterokinase present on the intestinal microvillus)

 Once activated, the trypsin activates other enzyme molecules.

 Trypsin hydrolyzes the bonds formed by carboxyl groups of Arg and Lys.

 Acute pancreatitis:

Premature activation of trypsinogen (pancreas)→

autodigestion of pancreatic cells → acute

pancreatitis

142) Chymotrypsin:

 Trypsin will act on chymotrypsinogen, so that the active site is formed. Thus, produces the

catalytic site. selective proteolysis.

3) Carboxypeptidases:

 Trypsin and chymotrypsin degrade the proteins into small peptides;

 these are hydrolyzed into dipeptides and tripeptides by carboxypeptidases present in the

pancreatic juice.

 They are Metalloenzymes requiring zinc.

15C. Intestinal digestion:

 The luminal surface of

intestinal epithelial cells

contains Amino- peptidases,

Carboxypeptidases and

dipeptidases which release

the N- terminal amino acids

successively.

16Absorption of A.A

 Small intestine.

 It is an energy requiring process.

 Carrier mediated systems.

 Glutathione (γ- glutamylcysteinylglycine) also plays an important role in the

absorption of amino acids.

17Amino Acids are used as..

 Metabolic fuel

 Substrate for protein synthesis

 Precursor

 Purine and pyrimidines

 Biologic amines

 Hormones

 Hem

 …….

1819Metabolic Fates

Of A.A:

 Body protein biosynthesis.

 Small peptide biosynthesis (GSH).

 Synthesis of non-protein nitrogenous (NPN) compounds (Creatine, Urea, Ammonia and Uric

Acid)

 Deamination & Transamination to synthesized a new amino acid or glucose or ketone bodies

or produce energy in starvation.

2021General metabolism of amino acids

1. Catabolic Pathway

 Catabolic Reactions.

 Dietary and body proteins are broken down to amino acids.

2. Anabolic pathway

 Anabolic Reaction:

 Amino acids are used for synthesis of body proteins

 Transdeamination Reaction:

1. Amino group of amino acids is transferred or removed to produce the carbon

skeleton (keto acid). The amino group is excreted as urea.

2. The carbon skeleton is used for synthesis of non- essential amino acids. It is also

used for gluconeogenesis or for complete oxidation.

22Transdeamination process

23 Amino acids are the major sources of

synthesis of Non-Protein Nitrogen (NPN)

compounds

 In Vertebrates & sharks:

 Urea

 In aquatic vertebrates:

 Ammonia (NH4

+)

 In birds, reptiles:

 Uric acid

24Metabolism OF AMINO ACIDS:

1. Remove of ammonia (NH3):

2. Fate of carbon-skeletons of amino acids

I. Deamination

3. Metabolism of ammonia

A. Oxidative deamination

1) glutamate dehydrogenase in mitochondria

2) amino acid oxidase in peroxisomes

B. Non-Oxidative (Direct deamination)

1. Deamination by dehydration (-H2O)

2. Deamination by desulfhydration (-H2S)

3. Threonine & lysine: directly deaminated

4. ….

II. Transamination (GPT & GOT)

III. Transdeamination

25Transamination

 Transaminases whose activities in serum are used as indices of liver damage catalyze

the reactions:

262728Biological significance of Transamination

 First step of catabolism:

Ammonia is removed, and rest of the AA is entered into catabolic pathway.

 Synthesis of non-essential amino acids:

all non-essential a.a could be synthesized by the body from keto acids available for other sources

29Kinds of Amino Acids

30A. Transamination

 Transamination is the exchange of amino group between amino acid and

another α-keto acid, forming a new alpha amino acid.

 The enzyme catalyzing the reaction

 Transaminases (amino transferases)

 Pyridoxal Phosphate ((PLP)(Vit B6 ) as a prosthetic group or Coenzyme.

 The reaction is readily reversible.

31Pyridoxal Phosphate (PLP)

 In the overall reaction, the amino acid transfers its amino group to pyridoxal phosphate and

then to the keto acid through formation of pyridoxamine phosphate as intermediate.

32Vitamin B6

 Pyridoxine (vegetables)

 Pyridoxal/Pyridoxamine (animal)

 Synthesized by microorganism and plants

 3 forms are active in the metabolism

 Coenzyme function (Pyridoxal phosphate)

 Crucial in the protein rich diet

33B. Trans-Deamination

 It means transamination followed by

oxidative deamination.

 All amino acids are first transaminated to

glutamate, which is then finally deaminated.

 They are found in the cytosol of the liver,

kidney, muscle and intestine cells.

 Glutamate dehydrogenase reaction is the

final reaction which removes the amino

group of all amino acids.

 It is one of the enzymes that can use both

NAD+ and NADP+ as electron acceptor.

34Two Common Amino Transferase Enzymes

1. Aspartate Amino Transferase (AST or SGOT)

35

352. Alanine Transaminase (ALT or SGPT)

36 Glutamate and glutamine have critical roles in nitrogen metabolism

 In hepatocytes amino goups are

transferred to α-ketoglutarate to

form glutamate.

 Glutamine is the major transport molecule for brain ammonia

 Alanine is the transporter for ammonia groups from muscle

37AST and ALT are clinically used as diagnostic markers

 AST : ~15-40 U/L (Cytoplasmic/Mitochondrial)

 ALT : ~ 10-40 U/L (only cytoplasmic)

 Mainly : Hepatic function tests

 Myocardial Infarction:

 Aspartate transaminase (AST)

 Alanine transaminase (ALT)

38Non-Oxidative Deamination

39Deamination

40Types of Deamination

1. Oxidative

 Takes place mostly in liver and kidney

 Provides ammonia for urea synthesis

 Amin groups of most a.a transferred to α-KG to produce glutamate

 Glutamate is oxidatively deaminated by GDH (Glutamate dehydrogenase )

 GDH is present only in liver mitochondria

 GDH can utilize NAD and NADP as coenzyme

412. Non-oxidative

 Remove ammonia without oxidation

 Examples:

1. Dehydrases: hydroxyl groups containing a.a(serine, threonine, homoserine

2. Disulfide bridges: sulfur containing a.a (cysteine)

3. Threonine & lysine: directly deaminated

Lyase

+

 Histidine Urocanate + NH4

Dehydratase

+

 Serine Pyruvate + NH4

Dehydratase

 Threonine & lysine +

α ketobutyrate + NH4

Deaminase

+

 Guanine Xanthine + NH4

+

Deaminase

 Adenosine Inosine + NH4

 Amino acid +

keto acid + NH4

(D ve L)

amino acid oxidase

4243Amino Acid Oxidation

44Summary of AA metabolisim

 Amino acids are not stored in the body

 Non-essential amino acids can be

 formed by transamination

 can be provided by eaten

 Transamination: Transfer of an amine group to keto acid.

 Deamination: Taking Ammonia out from amino acids

 Amine group of a.a transfers to α-ketoglutarat by transamination and forms glutamate

 Ammonia, which is an alanine in muscles and a glutamine in other tissues especially in brain,

is transferred to liver and then transfer to glutamate→ Glu transfer to mitochondria

 In the mitochondria, ammonia is released by deamination process which is catalyzed by

glutamate dehydrogenate enzyme. Ammonia and keto acids are produced as by-products of

oxidative deamination.

 Ammonia is converted to urea and excreted.

4546Aspartate- Glutamate Shuttle

47Formation of Ammonia

 The first step → remove the amino group as ammonia (NH4

+).

 Ammonia is highly toxic especially to the nervous system.

 Detoxification

 Ammonia → urea → excretion through urine.

4849why is Ammonia toxic ??

 Healthy adult blood levels : 11-60 mmol/L

 Increase ammonia (Hyperammonemia)

 Liver failure

 Brain is the primary target organ (Hepatic encephalopathy/hepatic coma)

 Causes of toxicity:

 Water insoluble: accumulate in our body

 Decrease in α-KG levels (combine with it)

 Decrease in ATP

 Decrease in NADH concentrations

 Edema related with glutamine osmolality

 Decrease of GABA concentrations

50Nitrogen Balance

 Nitrogen balance : difference between nitrogen consumed and excreted per day

 Nitrogen balance

N consumed = N excreted

 Negative nitrogen balance (Inadequate intake, fasting, diabetes)

N consumed < N excreted

 Positive nitrogen balance (Childhood, pregnancy, trauma, illness….)

N consumed > N excreted

 Important: Reveals liver or kidney damages

 Elevated in

 Amino Acid Catabolism

 Renal insufficiency

 Glutamate→ CPS I activation

 Decreased in → Hepatic Failure

51Amino groups

or ammonia

52Transport of Ammonia to Liver

 Ammonia (NH3 )is produced in most tissues – less than 1% is TOXIC especially to CNS

 Readily ionizes to ammonium ion NH4

+

 It is immediately removed from the circulation and detoxified to Urea in the LIVER.

 Transport forms of NH4

+ from peripheral tissues to LIVER:

1. Glutamine– ALL tissues (Brain)

2. Alanine– Muscle (GLUCOSE-ALANINE CYCLE)

 Ammonia (NH3 )

 ammonium ion NH4

+

 Since the enzymes of the urea cycle are present in the liver only, amino groups from

other tissues should be transported to the liver

53Transport of Ammonia to Liver

 CORI CYCLE: Utilization of lactate produced by anaerobic glycolysis in the muscles

 GLUCOSE-ALANINE CYCLE: Utilization of amino acid metabolites released from muscles54• Two mechanisms of transport:

1. The skeletal muscles export alanine synthesized from the transamination of

pyruvate (glucose catabolism) by glutamate

 The amino group donated by glutamate was obtained from the breakdown of

amino acids in the muscle

551. GLUCOSE-ALANINE CYCLE:

56Cori Cycle vs Alanine Cycle

5758

GLUCOSE-ALANINE CYCLE

581. 2. 2. Glutamate to Glutamine

α-KG accepts amino group which is catalyzed by glutamate dehydrogenase enzyme

and forms glutamate. Here NAD/NADP are oxidized.

Glutamate can accept another amino group through an ATP-dependent reaction

catalyzed by glutamine synthetase.

 Glutamine is used by most tissues to transport ammonium and travels to the liver.

59• Ammonia from extrahepatic tissues is transported to liver in the form of glutamine

• Enzyme: Glutamine synthetase

 the ammonium released by the deamination is used as a buffer (in the kidneys)

or enters the urea cycle in the liver;

602. Glutamate can accept another amino group through an ATP-dependent reaction

catalyzed by glutamine synthetase

 Glutamine is used by most tissues to transport ammonium

 Glutamine travels to the liver, kidneys and the intestine and is deamidated

 The ammonium released by the deamination is used as a buffer (in the

kidneys) or enters the urea cycle in the liver

 Glutamine can be used as an energy source by the intestine

61Transferring of glutamate from cytosol to mitochondria

62Urea Cycle

 Two molecules of ammonia

 Excreted as urea from kidney

 Non standart amino acids : Citrulline and ornithine Argininosuccinate ,

Arginine, Ornithine and Citrulline used

 Urea is added to cytosolic pool

 Five enzymes participate in the Urea cycle

 Six amino acid play role in the urea cycle

 ATP is used (2 ADP and 1 AMP(ATP to AMP))

 Associated with the Krebs cycle

 Start in mitochondria, finish in cytosol

 Three reactions are located in the cytosol

6364 The cycle is known as Krebs-Henseleit or as Ornithine cycle.

 The two nitrogen atoms of urea

1. One from ammonia

2. The other directly from aspartic acid.

65Urea cycle

Formation of

1. Carbamoyl Phosphate.

2. Citrulline.

3. Argininosuccinate.

4. Arginine.

5. Urea.

66o The two nitrogen atoms of urea enter the urea cycle as NH4

+ and as the amino N

of aspartate

1. The synthesis of carbamoyl phosphate

• The NH4

+ and HCO3

- (carbonyl C) that will be part of urea are incorporated first

into carbamoyl phosphate

 The cleavage of 2 ATP molecules is needed to form the high energy

carbamoyl phosphate

 Carbamoyl phosphate synthetase (CPS I) is a mitochondrial enzyme; the

cytosolic isozyme is involved in pyrimidine synthesis

• CPS I has an absolute requirement for the allosteric activator N-acetylglutamate

• This derivative of glutamate is synthesized from acetyl-CoA and glutamate so,

when cellular glutamate is high, signaling an excess of free amino acids due to

protein breakdown or dietary intake send to the cells

672. The formation of citrulline

• Carbamoyl phosphate reacts with ornitihine to give citrulline; catalyzed by

ornithine transcarbamoylase

3. The entry of the second N

• Citrulline leaves the mitochondria in exchange for the entry of ornithine

from the cytosol

• Citrulline reacts with aspartate producing argininosuccinate

• Argininosuccinate synthetase requires the splitting of ATP to AMP and PPi68+

+

Pi

ornithine trans-

carbamoylase

ATP AMP+ PPi

argininosuccinate

synthetase

694. The formation of arginine

• Argininosuccinate lyase produces arginine and fumarate

• The arginine produced by the urea cycle is enough for adults

• The carbons of fumarate are those that were obtained from aspartate

 Fumarate be changed to oxaloacetate by enzymes of the citric acid cycle

 The oxaloacetate will receive an amino group from glutamate and be

changed to aspartate; aspartate reenters the urea cycle

 The TCA and urea cycles constitute a bicycle: The Krebs bicycle

5. The production of urea and the regeneration of ornithine

• The action of arginase produces urea and ornithine

• Urea travels to the kidneys and excreted through the urine

70Argininosuccinase

+

71The urea cycle

72The Krebs bicycle

The stoichiometry of the urea cycle

NH4

++ CO2 + 3 ATP+ 2H2O + aspartate →urea + fumarate + 2 ADP + AMP+ 4Pi737475 Ammonia is excreted with H+ ions in the kidneys.

 This is important to acid-base balance.

76Ammonia

sources

77Ammonia in the liver is entered to urea cycle and excreted as urea

7879Urea molecule

80Intermediate molecules between urea and krebs cycle

81Ammonia metabolism of hepatocyte

 Ammonia (NH4

+) Sources:

 Amino acids

 Nucleic acids

 Amines

 Glutamate dehydrogenase

 Glutaminase

 Bacterial urease

 Urea urease 2NH3 + CO2

 Bacterial infections

82Control of Urea Cycle

 Diet rich in protein

 Starvation

Activities

 Allosteric control of CPS I (N-acetyl glutamate)

Increased Enzyme

 Arginino succinate synthetase is the rate limiting step

2 NH4

+ + HCO3

-

+ 3ATP4-

Urea+ 2ADP3- +4Pi2-

+ AMP2-

100 g protein intake 33 g Urea output

+ 5H+

83Genetic Disorders of Urea Cycle

Disorder Disease Characteristic

CPS I Deficiency Hyperammonemia %0-50 activity in infantil

period

N-acetyl glutamate

synthetase deficiency

— Carbamoyl glutamate

OTCase deficiency“Sex-linked”

Ornithinemia

[NH4

+]↑, [Ornithine ]↑,

[Orotic acid ]↑,

Argininosuccinate

synthetase deficiency

Citrullinemia [Citrulline]↑

Argininosuccinate lyase

deficiency

Argininosuccinic

aciduria

[Argininosuccinic acid]↑

Arginase deficiency Argininemia [Arg] ↑, excretion ↑

84Genetic disoders of amino acid metabolism

 Hyperammonemia and mental retardation

 Treatment:

1. Protein restriction

2. Essential amino acid keto analog

3. Removal of ammonia from circulation

 Benzoate and phenylacetate

 Benzoate + Glycine Hyppuric acid

Excretion with

urine

 Phenylacetate + Glutamine Phenylacetylglutamine

4. Replacement of deficient enzymes

5. In the deficiency of N-acetyl glutamate synthase : carbamoyl glutamate

6. OTCase, argininosuccinate synthetase and lyase deficiency : Arginine

supplementation

85Treatment of urea cycle disorders

86 The clinical symptoms associated with defect in urea cycle enzymes include

vomiting, lethargy, irritability, ataxia and mental re tardation

No Urine

orotate

Plasma

citrulline

Plasma

ornithine

Plasma

arginine

Plasma

ammonia

I Low Low Low Low High

II - High High Low High

III - - Low High High

IV High Low High Low High

V - High Low Low High

I. CPS I

II. Argininosuccinate synthatase

III. Arginase

IV. OTcase

V. Argininosuccinate lyase

87Urea Cycle

Enzymes Deficiencies

and related diseases

88Regulation of the urea cycle

 During starvation

 The activity of urea cycle enzymes is elevated

 Increased rate of protein catabolism.

 The major regulatory steps is catalyzed by CPS-I where the positive effector is

N-acetyl glutamate (NAG). 89