BYDr A.VEERENDRA KUMAR REDDY MBBS, MD

UREA CYCLE

Amino acid Catabolism

There are 20 different amino acid, they are monomeric constituents of proteins.

In mammalian tissues, α-NH2 group of amino acids, derived from the diet or breakdown tissue proteins, ultimately is converted first to NH3 and then urea and is excreted in the urine.

Urea is the end product of the amino acid catabolism.

Urea Cycle: Conversion of NH3 to urea for

excretion

Urea biosynthesis occurs in four stages:(1) Transamination(2) Oxidative deamination of glutamate (3) Ammonia transport (4) reactions of the urea cycle.

Also called as Krebs-Henseleit cycle or Ornithine cycle

First metabolic pathway to be elucidated(1932).

UREA CYCLE

Over all Nitrogen flow in Amino acid catabolism

Transamination: Transfer of amino group to α-ketoglutarate. There are several aminotransferases specific to different amino acids. In this step amino group from all the amino acids are transferred to a-ketoglutarate and they exist as glutamate.

Transaminases or aminotransferases require pyridoxal-5’-phophate PLP (vitamin B6 derivative)

PLP is very important cofactor for many enzymatic reactions.

1.Transamination

Transamination

2.Oxidative deamination:The amino group of glutamate is released as ammonia, regenerating α -ketoglutarate, by an enzyme glutamate dehydrogenase.

Glutamate dehydrogenase requires NAD+ or NADP+ as cofactor. This is the only enzyme known that has specificity for both type of cofactors.

This enzyme is allosterically inhibited by GTP and activated by ADP.

Over all Nitrogen flow in Amino acid catabolism

3.Transport of Ammonia

Sources of Ammonia:1. Ammonia is produced in the body from the different tissues by Amino acid catabolism.2. Purine and Pyramidine catabolism.3. The other source is from the dietary proteins and from urea present in fluids secreted into the GI tract.

Glutamine Synthetase

GLUTAMINE is the important plasma transport form of nitrogen from muscle.

Transport of excess ammonia by glutamine: Excess ammonia is toxic to animal tissues.

Glutamine synthetase catalyses the synthesis of glutamine by adding the ammonia to glutamate at the expense of ATP hydrolysis.

Glutamine is a non-toxic carrier of ammonia. It is transported to liver or kidney via blood.

In the liver & kidney, glutamine is reconverted to glutamate and ammonia by glutaminase. Ammonia is incorporated in urea cycle to form urea and then it will be excreted through kidneys.

Normal blood ammonia levels:15-45 micro gm/dL.

http://www.nlm.nih.gov/medlineplus/ency/article/003506.htm

Hyper Ammonaemia:1. Acquired hyperammonaemia: result of

Cirrhosis of Liver with development of collaterals circulation.

2. Inherited hyperammonaemia: results from genetic defects of the urea cycle enzymes.

NH3 Toxicity

Postulated mechanisms for toxicity of high ammonia:

1. High [NH3] would drive Glutamine Synthetase:

glutamate + ATP + NH3 glutamine + ADP + Pi

This would deplete glutamate – a neurotransmitter & precursor for synthesis of the neurotransmitter GABA.

2. Depletion of glutamate & high ammonia level would drive Glutamate Dehydrogenase reaction to reverse: glutamate + NAD(P)+ a-ketoglutarate + NAD(P)H + NH4

+

The resulting depletion of a-ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.

Features of Ammonia Toxicity:A peculiar flapping tremorSlurring of speechBlurring of visionComa and death.

Metabolic fate of NH3 in the body1. Mainly NH3 is converted to Urea(through

Urea cycle).2. Formation of Glutamine3. Amination of α-ketoacid to form α-amino

acid.

UREA CYCLE

Most terrestrial animals convert excess nitrogen to urea, prior to excreting it.

Urea is less toxic than ammonia.

The Urea Cycle occurs mainly in liver. First 2 reactions in mitochondria and rest in cytosol.

The 2 nitrogen atoms of urea enter the Urea Cycle as NH3 (produced mainly via Glutamate Dehydrogenase) and as the amino N of aspartate.

The NH3 and HCO3- (carbonyl C) that will be part of urea are

incorporated first into carbamoyl phosphate.

H 2 N C

O

N H 2

u r e a

H2N C OPO32

O

CH2

CH2

CH2

HC

COO

NH3+

NH3+

CH2

CH2

CH2

HC

COO

NH3+

NH

CO NH2

COO

CH2

HC

COO

NH2

CH2

CH2

CH2

HC

COO

NH3+

NH

C NH2+

COO

CH2

HC

COO

HN

AMP + PPi

ATP

CH2

CH2

CH2

HC

COO

NH3+

NH

C

NH2+H2N

COO

HC

CH

COO

C NH2H2N

O H2O

Pi

ornithine

urea

citrulline

aspartate

arginino- succinate

fumarate

arginine

carbamoyl phosphate

Urea Cycle

2

3

4

5

Urea CycleEnzymes in mitochondria: 1.Carbamoyl phosphate synthase I 2.OrnithineTrans-carbamylaseEnzymes in cytosol:3. Arginino- Succinate Synthase4. Arginino succinase

5. Arginase.

Step -1

Carbamoyl Phosphate Synthase (Type I) catalyzes a 3-step reaction, with carbonyl phosphate and carbamate intermediates.

Ammonia is the N input.

The reaction, which involves cleavage of 2 ~P bonds of ATP, is essentially irreversible.

H2N C OPO32

O

H2N C O

O

HO C

O

OPO32

HCO3

ATP

NH3

ADP ATP

Pi

ADP

carbonyl phosphate

carbamate

carbamoyl phosphate

Carbamoyl Phosphate Synthase I is the committed step of the Urea Cycle, and is subjected to regulation.

Carbamoyl Phosphate Synthase II: present in cytosol of liver cells and involved in synthesis of pyrimidines.

H 2N C O PO 32

O

HC O 3 + N H 3 + 2 A TP

+ 2 AD P + P i

C arbam oyl Phosphate Synthase

carbam oyl phosphate

Carbamoyl Phosphate Synthase 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.

H3N+ C COO

CH2

CH2

COO

H

glutamate (Glu)

NH

C COO

CH2

CH2

COO

H

CH3C

O

N-acetylglutamate

Step:2 (synthesis of Citrulline)Ornithine transcarbamoylase – mitochondrial Catalyses addition of ornithine to the carbonyl

group of Carbamoyl phosphate.

Step:3 (synthesis of Argininosuccinate)Argininosuccinate synthase – cytosolicATP -- AMP

Step:4 (Cleavage of Argininosuccinate)Argininosuccinase /Argininosuccinate lyase -

cytosolicArgininosuccinate-- Arginine + Fumarate

Step:5 ( Cleavage of Arginine to Ornithine and Urea)Enzyme: Arginase – cytosolic

ENERGETICS

2 ATPs are utilized for the synthesis of carbamoyl phosphate.

1 ATP is converted to AMP and Ppi to produce Arginosuccinate which is equals to 2 ATP

Net utilization = 4 ATP

NH4+ + CO2 + aspartate + 3 ATP --->

urea + fumarate + 2 ADP + AMP + 4 Pi

Regulation of urea cycle:

1.The reaction catalyzed by carbamoyl phosphate synthase I is the rate limiting reaction and committed step of Urea cycle

It is allosterically activated by N-acetylglutamate(NAG).

2. Consumption of protein rich meal increases the levels of NAG in liver, leading to enhanced urea synthesis.

3. Carbamoyl phosphate synthase I and glutamate dehydrogenase are located in mitochondria. They coordinate each other in the formation of NH3, and its utilization for the synthesis of carbamoyl phosphate.

The remaining enzymes of urea cycle are mostly controlled by the concentrations of their respective substrates.

Interaction of Urea Cycle and Citric Acid Cycle via Aspartate-Argininosuccinate shunt

UREA CYCLE DISORDERSS.No Disorder Enzyme involved

1. Hyperammonemia type I Carbamoyl phosphate synthase I

2. Hyperammonemia type II Ornithine transcarbamoylase

3. Citrllinemia Arginosuccinate synthase

4. Arginosuccinicaciduria Arginosuccinase

5. Hyperargininemia Arginase

Hereditary deficiency of any of the Urea Cycle enzymes leads to hyperammonemia - elevated [ammonia] in blood.

Elevated ammonia is toxic, especially to the brain.

Other metabolites of urea cycle accumulate depending on specific enzyme defect.

The clinical symptoms: Vomiting, lethargy, irritability, ataxia, and metal retardation.

If not treated immediately after birth, severe mental retardation results.

Total lack of any Urea Cycle enzyme is lethal.

Hyperammonemia type I:Familial disorder, produces hyperammonemia

and symptoms of ammonia toxicity.

Hyperammonemia type II:X linked, produces symptoms of ammonia

toxicity.Increased levels of glutamine (glutamine

synthesis enhanced in response to elevated NH3)

Postulated mechanisms for toxicity of high [ammonia]:

1. High [NH3] would drive Glutamine Synthase:

glutamate + ATP + NH3 glutamine + ADP + Pi

This would deplete glutamate – a neurotransmitter & precursor for synthesis of the neurotransmitter GABA.

2. Depletion of glutamate & high ammonia level would drive Glutamate Dehydrogenase reaction to reverse: glutamate + NAD(P)+ a-ketoglutarate + NAD(P)H + NH4

+

The resulting depletion of a-ketoglutarate, an essential Krebs Cycle intermediate, could impair energy metabolism in the brain.

Possible therapies for the patients with defect in urea cycle:

1. Defined diet containing just the minimum amount of essential amino acids.

2. Feeding the patients with Benzoate or phenylacectate: These compound react with glycine and glutamine respectively forming non-toxic compounds that are excreted in urine. Thus the body runs low in glycine and glutamine and starts synthesizing these AA using the ammonia available in system. Thus clearing the system of excess ammonia.

3. In the patients with N-acetylglutamate synthase deficiency, Carbamoyl glutamate can act as activator of carbamoyl phosphate synthase.

4.Liver transplantation has also been used, since liver is the organ that carries out Urea Cycle.

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