Unit 6: Metabolism III - Subjective Questions

Transamination is the reversible transfer of an amino group () from an amino acid to an -keto acid, resulting in the formation of a new amino acid and a new -keto acid. It is catalyzed by enzymes called transaminases (aminotransferases).

General reaction:

Mechanism:

• The coenzyme pyridoxal phosphate (PLP), derived from vitamin , is essential.
• PLP forms a Schiff base (aldimine) with the amino group of the amino acid.
• The amino group is transferred to PLP forming pyridoxamine phosphate (PMP), releasing the corresponding -keto acid.
• PMP then transfers the amino group to a new -keto acid, regenerating PLP.

Example (Alanine transaminase, ALT):

Significance:

• Central role in amino acid metabolism.
• Channels amino groups toward glutamate for eventual disposal.
• Serum ALT and AST are important diagnostic markers of liver and heart damage.

Oxidative deamination is the removal of an amino group from an amino acid as ammonia, coupled with oxidation. The most important reaction is catalyzed by glutamate dehydrogenase (GDH) in the mitochondria of liver cells.

Reaction:

Key features:

• Uses either or as coenzyme.
• It is reversible, allowing both catabolism and biosynthesis.
• Allosterically activated by ADP and GDP (low energy) and inhibited by ATP and GTP (high energy).

Importance:

• It is the link between transamination and ammonia release. Amino groups collected on glutamate via transamination are liberated as free ammonia here.
• The liberated enters the urea cycle for detoxification.
• Regenerates -ketoglutarate to continue collecting amino groups.
• Provides a bridge between amino acid metabolism and the citric acid cycle.

Amino acids are classified based on the metabolic fate of their carbon skeletons.

Both glucogenic and ketogenic:

• Isoleucine, Phenylalanine, Tyrosine, Tryptophan, Threonine.

Purely ketogenic: Only Leucine and Lysine.

Significance:

• Glucogenic amino acids maintain blood glucose during fasting/starvation.
• Ketogenic amino acids contribute to energy via ketone bodies, especially important for the brain during prolonged starvation.

The urea cycle (Krebs-Henseleit cycle) is the pathway by which toxic ammonia is converted to urea for excretion. It occurs partly in the mitochondria and partly in the cytosol of liver cells.

Steps:

1. Formation of carbamoyl phosphate (mitochondria):

Enzyme: Carbamoyl phosphate synthetase I (CPS I) (activated by N-acetylglutamate).

2. Formation of citrulline (mitochondria):

Enzyme: Ornithine transcarbamoylase. Citrulline is transported to cytosol.

3. Formation of argininosuccinate (cytosol):

Enzyme: Argininosuccinate synthetase.

4. Cleavage to arginine and fumarate (cytosol):

Enzyme: Argininosuccinate lyase.

5. Hydrolysis of arginine (cytosol):

Enzyme: Arginase. Ornithine is regenerated and re-enters the mitochondria.

Overall reaction:

Thus, one nitrogen comes from ammonia and the other from aspartate.

Significance of the urea cycle:

• Detoxification of ammonia: Ammonia is highly toxic, especially to the brain. The urea cycle converts it to non-toxic, water-soluble urea.
• Nitrogen excretion: Urea is the major end product of protein/amino acid nitrogen metabolism in humans; excreted via urine.
• Maintains nitrogen balance in the body.
• Occurs mainly in the liver, the primary site of ammonia disposal.

Connection with the citric acid cycle (Krebs bi-cycle):

• The urea cycle produces fumarate (step 4), which is a TCA cycle intermediate.
• Fumarate is converted to malate and then to oxaloacetate (OAA) in the TCA cycle.
• OAA can be transaminated to aspartate, which re-enters the urea cycle to supply the second nitrogen.
• This interlinking is called the "Krebs bi-cycle" because the two cycles are metabolically connected through fumarate and aspartate.

Energetic aspect:

• 4 high-energy phosphate bonds are consumed per urea molecule, but the fumarate/malate/OAA route can generate NADH, partially offsetting the cost.

De novo purine synthesis builds the purine ring stepwise on a ribose-5-phosphate backbone, forming inosine monophosphate (IMP) first.

Sources of purine ring atoms:

• N1 from aspartate
• C2, C8 from formate (-formyl-THF)
• N3, N9 from glutamine
• C4, C5, N7 from glycine
• C6 from

Key steps:

1. Ribose-5-phosphate + ATP PRPP (phosphoribosyl pyrophosphate) by PRPP synthetase.
2. PRPP + glutamine 5-phosphoribosylamine by glutamine-PRPP amidotransferase (committed, rate-limiting step).
3. A series of ~10 reactions add glycine, formyl groups, glutamine amide, , and aspartate to build the ring, forming IMP.

Conversion of IMP:

• IMP AMP (requires aspartate and GTP)
• IMP GMP (requires glutamine and ATP)

Regulation:

• Glutamine-PRPP amidotransferase is inhibited by AMP, GMP, IMP (feedback inhibition).
• AMP synthesis requires GTP and GMP synthesis requires ATP, ensuring balanced production of both nucleotides.
• PRPP synthetase is inhibited by ADP and GDP.

In pyrimidine biosynthesis, the ring is synthesized first, and then attached to ribose-5-phosphate, unlike purine synthesis where the ring is built on the sugar.

Key steps:

1. Carbamoyl phosphate formation from glutamine + + 2ATP by carbamoyl phosphate synthetase II (CPS II) in the cytosol (rate-limiting step).
2. Carbamoyl phosphate + aspartate carbamoyl aspartate by aspartate transcarbamoylase (ATCase).
3. Ring closure dihydroorotate by dihydroorotase.
4. Oxidation orotate by dihydroorotate dehydrogenase.
5. Orotate + PRPP orotidine monophosphate (OMP).
6. Decarboxylation of OMP UMP (uridine monophosphate).
7. UMP UDP UTP CTP (amination by CTP synthetase using glutamine).

Differences from purine synthesis:

The salvage pathway reutilizes free purine and pyrimidine bases (released during nucleic acid degradation) to synthesize nucleotides, avoiding the energetically expensive de novo route.

Purine salvage enzymes:

• Adenine phosphoribosyltransferase (APRT):
  
• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT):
  
  

Advantages:

• Energy efficient (de novo synthesis needs many ATP).
• Important in tissues like the brain that have low de novo capacity.

Clinical significance:

• Lesch-Nyhan syndrome: Complete deficiency of HGPRT leads to excess purine degradation, hyperuricemia, gout, mental retardation, and self-mutilation.
• Salvage pathway is also targeted by chemotherapy drugs and immunosuppressants.

Purine catabolism in humans ends with the formation of uric acid, which is excreted in urine.

Pathway:

1. AMP (deamination by AMP deaminase) IMP; or AMP adenosine inosine.
2. IMP / GMP are dephosphorylated to nucleosides (inosine, guanosine) by 5'-nucleotidase.
3. Nucleosides are cleaved by purine nucleoside phosphorylase (PNP) to free bases:
   • Inosine hypoxanthine
   • Guanosine guanine
4. Hypoxanthine Xanthine.
5. Guanine Xanthine.
6. Xanthine Uric acid.

Overall:

Clinical significance:

• Overproduction or under-excretion of uric acid causes gout (deposition of monosodium urate crystals in joints).
• Allopurinol, a xanthine oxidase inhibitor, is used to treat gout.

Pyrimidine catabolism produces highly soluble products, unlike the relatively insoluble uric acid from purines.

Pathway:

1. Pyrimidine nucleotides are dephosphorylated and cleaved to free bases cytosine, uracil, thymine.
2. Cytosine is deaminated to uracil.
3. Uracil dihydrouracil -ureidopropionate -alanine + + .
4. Thymine dihydrothymine -ureidoisobutyrate -aminoisobutyrate + + .

End products:

• -alanine and -aminoisobutyrate (excreted or further metabolized).
• Ammonia (enters urea cycle) and .

Differences from purine degradation:

Pyridoxal phosphate (PLP) is the active coenzyme form of vitamin (pyridoxine) and is essential for transamination.

Role:

• Acts as an amino group carrier during transamination.
• Serves as an electron sink, stabilizing the carbanion intermediate.

Mechanism:

1. In the resting enzyme, PLP is bound to a lysine residue through a Schiff base (internal aldimine).
2. The amino acid substrate displaces lysine, forming an external aldimine (Schiff base) with PLP.
3. Removal of the -hydrogen forms a quinonoid intermediate, stabilized by the pyridine ring acting as an electron sink.
4. Reprotonation forms a ketimine, which is hydrolyzed to release the -keto acid, leaving PLP as pyridoxamine phosphate (PMP).
5. In the second half-reaction, PMP transfers the amino group to a new -keto acid, regenerating PLP and forming a new amino acid.

Significance:

• This ping-pong (bi-bi) mechanism allows reversible amino group transfer.
• PLP is also involved in decarboxylation, deamination, and racemization reactions of amino acids.

Deficiencies in any of the five urea cycle enzymes lead to hyperammonemia due to failure of ammonia detoxification.

Major disorders:

• CPS I deficiency: Severe hyperammonemia, present early in life.
• Ornithine transcarbamoylase (OTC) deficiency: Most common; X-linked; causes elevated ammonia and orotic acid.
• Citrullinemia: Argininosuccinate synthetase deficiency; citrulline accumulates.
• Argininosuccinic aciduria: Argininosuccinate lyase deficiency.
• Hyperargininemia (Argininemia): Arginase deficiency.

Clinical consequences of hyperammonemia:

• Ammonia is neurotoxic; it depletes -ketoglutarate (by forming glutamate/glutamine), impairing the TCA cycle in the brain.
• Symptoms: lethargy, vomiting, tremors, slurred speech, cerebral edema, coma, and death if untreated.
• Elevated glutamine causes osmotic swelling of astrocytes.

Management:

• Low-protein diet.
• Drugs like sodium benzoate and phenylbutyrate to provide alternative nitrogen excretion routes.
• Supplementation of arginine/citrulline depending on the defect.

After removal of the amino group, the remaining carbon skeletons of the 20 standard amino acids are converted into seven metabolic intermediates that enter central metabolism.

The seven products:

1. Pyruvate – Alanine, Glycine, Serine, Cysteine, Threonine, Tryptophan
2. Acetyl-CoA – Leucine, Lysine, Isoleucine, Tryptophan, Phenylalanine, Tyrosine, Threonine
3. Acetoacetyl-CoA / Acetoacetate – Leucine, Lysine, Phenylalanine, Tyrosine, Tryptophan
4. -Ketoglutarate – Glutamate, Glutamine, Proline, Arginine, Histidine
5. Succinyl-CoA – Methionine, Valine, Isoleucine, Threonine
6. Fumarate – Phenylalanine, Tyrosine, Aspartate
7. Oxaloacetate – Aspartate, Asparagine

Fate:

• Those forming pyruvate or TCA intermediates (-KG, succinyl-CoA, fumarate, OAA) are glucogenic.
• Those forming acetyl-CoA or acetoacetyl-CoA are ketogenic.

This links amino acid catabolism to gluconeogenesis, ketogenesis, and energy production via the citric acid cycle.

Free ammonia is toxic, so it must be transported in non-toxic forms to the liver for conversion to urea.

1. Glutamine transport (most tissues):

• Ammonia combines with glutamate to form glutamine via glutamine synthetase:
  
• Glutamine travels in blood to the liver and kidney.
• Glutaminase releases ammonia in the liver:
  

2. Glucose-Alanine cycle (muscle):

• In muscle, amino groups are transferred to pyruvate forming alanine (via transamination).
• Alanine travels to the liver where it is transaminated back to pyruvate, releasing the amino group.
• Pyruvate is used for gluconeogenesis; glucose returns to muscle.
• Ammonia enters the urea cycle.

Significance:

• Safely transports nitrogen while also shuttling carbon for gluconeogenesis.
• Links muscle protein catabolism to hepatic glucose production during fasting/exercise.

The urea cycle is regulated to match the rate of ammonia disposal with dietary protein intake and metabolic state.

Short-term (allosteric) regulation:

• N-acetylglutamate (NAG) is an essential allosteric activator of carbamoyl phosphate synthetase I (CPS I), the rate-limiting enzyme.
• NAG is synthesized from acetyl-CoA and glutamate by N-acetylglutamate synthase, which is activated by arginine.
• High protein/amino acid load raises glutamate and arginine, increasing NAG, thereby stimulating the urea cycle.

Long-term (substrate-level & induction) regulation:

• On a high-protein diet or during starvation (increased protein breakdown), the synthesis of all five urea cycle enzymes is induced (increased gene expression).
• On a low-protein diet, enzyme levels decrease.

Substrate availability:

• Increased ammonia and amino acid supply drives higher flux through the cycle.

Thus, regulation ensures nitrogen is efficiently excreted without wasting energy when protein intake is low.

Summary:

• De novo builds nucleotides from scratch, essential for growth and replication.
• Salvage conserves energy by reusing bases, crucial in tissues that cannot afford full de novo synthesis.

Deoxyribonucleotides (dNTPs) required for DNA synthesis are formed by the reduction of ribonucleotides at the 2'-carbon of the ribose sugar.

Key enzyme: Ribonucleotide reductase (RNR)

• Catalyzes reduction of ribonucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs):
  
• Substrates: ADP, GDP, CDP, UDP dADP, dGDP, dCDP, dUDP.

Reducing equivalents:

• Supplied by thioredoxin (or glutaredoxin), which is reduced by thioredoxin reductase using NADPH.

Regulation:

• RNR has allosteric activity sites and specificity sites that maintain balanced pools of all four dNTPs.
• ATP activates; dATP inhibits overall activity.

Formation of dTMP:

• dUMP is methylated to dTMP by thymidylate synthase, using -methylene-THF.
• This step is a target of anticancer drugs (5-fluorouracil, methotrexate).

Significance: RNR provides the sole route for de novo dNTP synthesis, essential for DNA replication and repair.

Gout is a metabolic disorder characterized by hyperuricemia (elevated blood uric acid) leading to deposition of monosodium urate crystals in joints and tissues, causing painful arthritis.

Biochemical basis:

• Uric acid is the end product of purine catabolism in humans.
• Gout results from either:
  • Overproduction of uric acid (e.g., PRPP synthetase overactivity, HGPRT deficiency, increased purine turnover), or
  • Under-excretion of uric acid by the kidneys.
• Uric acid has low solubility; excess leads to crystal deposition, especially in the big toe (podagra), causing inflammation.

Associated conditions:

• Lesch-Nyhan syndrome (HGPRT deficiency) causes severe hyperuricemia.
• Kidney stones (urate calculi) may form.

Treatment:

• Allopurinol: inhibits xanthine oxidase, reducing uric acid production (xanthine and hypoxanthine are more soluble).
• Colchicine and NSAIDs: reduce acute inflammation.
• Uricosuric agents (e.g., probenecid): increase renal excretion.
• Dietary control: reduce purine-rich foods and alcohol.

Comparison of purine and pyrimidine biosynthesis:

Feedback regulation of purine synthesis:

• Glutamine-PRPP amidotransferase inhibited by AMP, GMP, IMP.
• IMP AMP requires GTP; IMP GMP requires ATP, ensuring cross-regulation and balance.
• PRPP synthetase inhibited by ADP, GDP.

Feedback regulation of pyrimidine synthesis:

• In bacteria, ATCase is inhibited by CTP and activated by ATP.
• In mammals, CPS II is inhibited by UTP and activated by PRPP and ATP.

Coordination:

• ATP and PRPP levels coordinate both pathways so that purine and pyrimidine nucleotides are produced in balanced amounts for nucleic acid synthesis.

Ammonia () is highly toxic, particularly to the central nervous system, and its accumulation (hyperammonemia) can cause encephalopathy, coma, and death.

Biochemical mechanisms of toxicity:

1. Depletion of -ketoglutarate:

• Excess ammonia drives the glutamate dehydrogenase reaction in reverse:
  
• This depletes -ketoglutarate, a key TCA cycle intermediate, impairing energy (ATP) production in the brain.

2. Accumulation of glutamine:

• Ammonia combines with glutamate to form glutamine in astrocytes:
  
• High glutamine causes osmotic swelling of astrocytes, leading to cerebral edema and increased intracranial pressure.

3. Neurotransmitter imbalance:

• Depletion of glutamate (an excitatory neurotransmitter) and altered GABA levels disturb neurotransmission.

Consequences:

• Impaired energy metabolism, cerebral edema, and neurotransmitter disturbances cause hepatic encephalopathy, seen in liver failure and urea cycle disorders.

Prevention: Efficient conversion of ammonia to urea (liver) and glutamine (peripheral tissues) keeps blood ammonia low.