dr. saidunnisa professor of biochemistry transamination, deamination and ammonia metabolism

Dr. SaidunnisaProfessor of Biochemistry

Transamination, Deamination and Ammonia Metabolism.

Learning objectives• At the end of session the student shall be able to:

1. Define transamination and deamination, mechanism of action with enzymes and co enzymes involved with suitable examples and its clinical application and its limitations.

2. Clinical importance of Glucose –alanine cycle.

3. Explain the processes of oxidative and non-oxidative deamination-enzymes and coenzymes required for the reaction.

4. Explain the role of Glutamine and Glucose-Alanine cycle in transporting amino nitrogen to the liver for urea synthesis.

5. Explain the biochemical basis for ammonia toxicity.

General aspects of amino acid metabolism

• Amino acids undergo certain common reactions like transamination followed by deamination to liberate ammonia which is converted to urea.

• The carbon skeletons of amino acids is first converted to ketoacids by transamination which meet one or more of the following fates:

1. Utilized to generate energy.2. Used for the synthesis of glucose.3. Diverted for the formation of fat or ketone bodies.4. Involved in production of non-essential aminoacids.

Overview of amino acid metabolism

1. Transamination2. Deamination3. Ammonia metabolism4. Urea synthesis

TRANSAMINATION

• Definition: is a reversible reaction in which alpha-NH2 group of one amino acid is transferred to a alpha-Keto acid resulting in the formation of a new amino acid and a new Keto acid

• The processes represents only intra molecular transfer of amino group no free ammonia liberated.

• Donor amino acid (1) thus becomes a new Keto acid (1) after losing the alpha-amino, and the recipient Keto acid (2) becomes a new amino acid (2) after receiving the amino group.

Transamination (ALT/AST)

Salient Features of Transamination

1. Reversible reaction

2. Site: takes place principally in liver, heart and brain

3. Enzymes: concerned are transaminases or aminotransferases named after the amino acid that serves as the amino group donor

3. Coenzyme for the reaction: PLP (pyridoxal phosphate B6) amino acid reacts with enzyme bound PLP to form enzyme bound complex i.e. Schiff base

4. Amino acid undergo transamination to finally concentrate nitrogen in glutamate which acts as a collecting point for the alpha-amino groups

5. Glutamate is the only amino acid that undergoes oxidative deamination to a significant extent to liberate free NH3 for urea synthesis.

6. Presence of these enzymes in cardiac muscle and liver cells makes them useful markers of tissue damage.

7. Concentration of these enzymes in blood increase after heart attack; where damaged heart muscle leaks its intracellular contents

8. Amino acids which do not take part in transamination are lysine, Threonine, Proline and Hydroxy Proline.

9. Transamination is responsible for the synthesis of Non essential amino acids.

DEAMINATION

• Removal of α-amino group from the amino acid as NH3 is deamination which is used for urea synthesis.

Two Types:• Oxidative deamination• Non oxidative deamination

Oxidative Deamination

• Definition: Is liberation of free ammonia from the amino group of amino acid coupled with oxidation.

• Site: takes place mostly in mitochondria of liver and kidney

• Purpose oxidative deamination is to provide NH3 for urea synthesis and α-Keto acids for a variety of reactions including energy generations.

Role of glutamate Dehydrogenase(GLDH)

• Glutamate acts as a collecting point for the α amino groups.

• Glutamate undergoes oxidative deamination catalyzed by glutamate Dehydrogenase which is a zinc containing enzyme to liberate ammonia.

• This enzyme is unique in that it can utilize NAD or NADP.

Regulation

• GTP and ATP are Allosteric inhibitors

• GDP and ADP are Allosteric activators

• GLDH can catalyze the reverse reaction also i.e amination of α-KG to form glutamate. This reaction requires NADPH as coenzyme.

Glutamate plays a key role

• The three mammalian enzymes that can fix ammonia into organic molecules are:

• GLDH• Glutamine Synthetase• Carbamoyl phosphate

Synthetase-I

Amino acidsα-ketoglutarate

Glutamate

α keto acidsTRANSAMINATION

NH4+

Other sources

GDHOxidative deamination

Urea cycleUrea

Non-oxidative deamination

• Definition: Some of the amino acids can be deaminated to liberate NH3 with out undergoing oxidation.

• These reactions do contribute to NH3 formation, but again they do not fulfill a major role in NH3 formation.

Non-oxidative deaminationTypes

• Three types:

1. Amino acid dehydrases

2. Amino acid desulfhydrases

3. Deamination of Histidine

Amino acid dehydrases

• Hydroxy amino acids eg serine, Threonine, and homoserine are deaminated by specific amino acid dehydrases which contain PLP as coenzyme .

• The enzymes catalyze a primary dehydration followed by spontaneous deamination.

• Serine/Threonine/ Dehydratase----------Ketoacids Homoserine +NH3

Amino acid desulfhydrases

• S-containing amino acids eg cysteine and Homocysteine are deaminated by desulhydration (removal of H2S) forming an imino acid which is spontaneously hydrolyzed to ketoacid( pyruvic acid)

• Cysteine- Desulfhydrases Pyruvate+H2S+

NH3

Deamination of Histidine

• Histidine is deaminated by the specific enzyme histidase to form NH3 and urocanic acid.

• Histidine- Histidase urocanate+NH3

Role of alanine and glutamine in transporting amino acid nitrogen to the liver.

• Alanine and glutamine are the major carriers of nitrogen in the blood.

• Alanine is primarily exported by the muscle.

• Because the muscle is metabolizing glucose through the glycolysis, pyruvate is available in the muscle.

• The pyruvate is transaminated by glutamate to form alanine, which travels to the liver.

Glucose-alanine cycle.

• On arriving at the liver, alanine is transaminated to pyruvate, and the nitrogen will be used for urea synthesis.

• The pyruvate formed is used for Gluconeogenesis and the glucose exported to the muscle for use as energy.

• This cycle of moving carbons and nitrogen's between the muscle and liver is known as the Glucose-alanine cycle.

Glucose-Alanine Cycle.

• Purpose of the glucose-alanine cycle:

• To release energy from pyruvate.

• To transport the amino nitrogen from the muscles to the liver.

• From the liver to the muscles, you get only glucose, not nitrogen, as it is removed by the urea cycle.

• Nitrogen transport is either in the form of alanine or in the form of glutamate.

Role of glutamine

• Under conditions of rapid amino acid degradation within tissues, such that ammonia levels increase, the glutamate that has been formed from transamination reactions will accept another nitrogen molecule to form glutamine.

• Glutamine is synthesized from glutamate by the fixation of ammonia, requiring energy ATP and the enzyme glutamine Synthetase which is a cytoplasmic enzyme found in all cells.

• The glutamine travels to the liver, kidney, or intestines, where glutaminase will remove the amide nitrogen to form glutamate plus ammonia.

• In the kidneys, the release of ammonia, and the formation of ammonium ion serves to form salts with metabolic acids in the urine.

• In the intestine, the glutamine is used as a fuel.

• In the liver, the ammonia is used for urea biosynthesis.

TCA cycle in transamination

Metabolism of Ammonia

• Ammonia is constantly being liberated in the metabolism of amino acids.

• At physiological pH ammonia exists as ammonium (NH4+)

ion. • This metabolism can be studied under following stages:1. Formation of Ammonia2. Transport and Storage of NH3

3. Functions of ammonia4. Disposal of Ammonia5. Toxicity of Ammonia

1. Formation of Ammonia

• The production of ammonia occurs from the AA (Transamination and Deamination)

2. Transport and Storage of NH3

• The transport of ammonia mostly occurs in the form of glutamine or Alanine and not as free ammonia.

• Alanine is important for NH3 transport from muscle to liver by glucose – alanine cycle.

• Role of glutamine: Glutamine is a store house of NH3 it servers as both transport and storage form.

• Ammonia is removed from the brain predominantly as glutamine.

3. Functions of ammonia

• Ammonia is not a waste product of nitrogen metabolism it is involved in synthesis of various compounds like

• NEAA• Purines• Pyrimidines• Amino sugars• Maintaining acid base balance.

4. Disposal of Ammonia

• The organisms during evolution have developed different mechanisms for the disposal of ammonia from the body.

• Ammoniotelic: The aquatic animals dispose off NH3 into surrounding water.

• Uricotelic: Ammonia is converted to uric acid eg reptiles and birds

• Ureotelic: The mammals including man convert NH3 to urea which is nontoxic, soluble and easily excreted.

5. Toxicity of Ammonia

• Even marginal elevation of ammonia (normal levels: 10-20ug/l) concentration is harmful to the brain.

• Ammonia when accumulates in the brain results in slurring of speech, blurring of vision, tremors and leads to coma and death.

• Hyperammonemia: can be genetic or acquired.• Genetic: due to deficiency of any one of five

enzymes of urea cycle• Acquired: Hepatitis and alcoholism.

Explanation of NH3 toxicity

• The reaction catalyzed by GLDH probably explains:

• Accumulation of NH3 shifts the equilibrium with more glutamate formation, hence more utilization of Alpha KG which is an intermediate of TCA cycle and it is depleted and TCA cycle is impaired.

• The net result is that ATP production by the brain is reduced.

• The toxic effects of NH3 on brain are therefore due to impairment in ATP formation.

• Why PLP deficiency causes microcytic hypochromic anemia?