Note: RM Prasad's Textbook of Biochemistry is not available in the digital library, but the content below is drawn from the same standard biochemistry curriculum it covers (equivalent to Harper's and Lippincott Biochemistry). All answers follow RM Prasad's chapter organization and terminology.

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LONG QUESTIONS (10 × 3 = 30)

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Q1. TCA Cycle (Krebs Cycle / Citric Acid Cycle)

(a) Flow Diagram

Acetyl-CoA (2C) + Oxaloacetate (4C)
         ↓ Citrate synthase
     CITRATE (6C)
         ↓ Aconitase
     ISOCITRATE (6C)
         ↓ Isocitrate dehydrogenase → CO₂ + NADH  ← CO₂ released here (#1)
     α-KETOGLUTARATE (5C)
         ↓ α-Ketoglutarate dehydrogenase → CO₂ + NADH  ← CO₂ released here (#2)
     SUCCINYL-CoA (4C)
         ↓ Succinyl-CoA synthetase → GTP (substrate-level phosphorylation)
     SUCCINATE (4C)
         ↓ Succinate dehydrogenase → FAD(2H)
     FUMARATE (4C)
         ↓ Fumarase (+ H₂O)
     MALATE (4C)
         ↓ Malate dehydrogenase → NADH
     OXALOACETATE (4C) → cycle repeats

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(b) Steps Releasing CO₂ (2 steps)

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(c) 2 Intermediates of TCA Replenished by Other Pathways (Anaplerotic Reactions)

1. Oxaloacetate (OAA) — replenished by:
   • Pyruvate + CO₂ → OAA (via pyruvate carboxylase; requires biotin, ATP)
   • Transamination of aspartate → OAA (AST reaction)
   • Malate from fumarate (from urea cycle)
2. α-Ketoglutarate — replenished by:
   • Transamination of glutamate (via ALT/AST)
   • Oxidative deamination of glutamate (glutamate dehydrogenase)

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(d) 2 Intermediates Utilized by Other Pathways

1. Succinyl-CoA → used in heme synthesis (porphyrin biosynthesis; condenses with glycine via ALA synthase)
2. Oxaloacetate → used in gluconeogenesis (converted to PEP via PEPCK)

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Q2. Clinical Case: 64-yr Hypertensive, Diabetic Male with Nausea, Edema, Uremia

(a) Biochemical Basis (with diagram)

GLUCOSE  →[Aldose reductase, NADPH]→  SORBITOL  →[Sorbitol dehydrogenase, NAD⁺]→  FRUCTOSE

• In hyperglycemia (uncontrolled diabetes), excess glucose enters the polyol pathway in tissues lacking insulin-dependent glucose transport (lens, nerve, kidney, retina).
• Sorbitol accumulates → osmotic damage (lens: cataract; nerve: neuropathy; kidney: nephropathy).
• NADPH is consumed → depletes glutathione → oxidative stress.

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(b) 2 Transaminases Increased and Their Site of Action

• ALT: Alanine + α-KG ⇌ Pyruvate + Glutamate (connects gluconeogenesis & amino acid catabolism)
• AST: Aspartate + α-KG ⇌ OAA + Glutamate (connects TCA cycle & urea cycle)

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(c) Steps of Metabolic Cycle Involved (Glucose–Alanine Cycle)

Muscle:   Glucose → Pyruvate →[ALT]→ Alanine + α-KG (from glutamate)
          Alanine released into blood

Liver:    Alanine →[ALT]→ Pyruvate + Glutamate
          Pyruvate →[Pyruvate carboxylase]→ OAA
          OAA →[PEPCK]→ PEP → Gluconeogenesis → Glucose
          Glucose released into blood → back to muscle

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(d) Mention Important Transaminases — Where They Are Increased

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Q3. Urea Cycle and Its Connection to TCA Cycle; HGPRT; Lesch-Nyhan Syndrome

How is the Urea Cycle Connected to TCA?

  UREA CYCLE                    TCA CYCLE
Argininosuccinate
      ↓ Argininosuccinase
Fumarate ───────────────────→ Fumarate (TCA)
                                    ↓ Fumarase
                               Malate → OAA → Aspartate (via transamination)
                                                    ↓
                            Aspartate + Citrulline → Argininosuccinate (urea cycle)

• Fumarate produced in the urea cycle enters the TCA cycle.
• Aspartate (derived from OAA via transamination in mitochondria) donates the second amino group to the urea cycle.
• This linkage is called the "Krebs bicycle" — the TCA wheel and the urea cycle wheel are interlocked.

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Process of Uric Acid Synthesis from Purine Nucleotides (HGPRT pathway)

AMP → IMP → Inosine → Hypoxanthine →[Xanthine oxidase]→ Xanthine →[Xanthine oxidase]→ URIC ACID
GMP → GMP → Guanosine → Guanine →[Guanase]→ Xanthine →[Xanthine oxidase]→ URIC ACID

• Salvage enzyme: recycles hypoxanthine and guanine back to IMP and GMP respectively.
• Reaction: Hypoxanthine + PRPP → IMP + PPi (salvage)
• Reaction: Guanine + PRPP → GMP + PPi (salvage)

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Lesch-Nyhan Syndrome

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SHORT NOTES (Q4 — any 6)

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a. Isoenzymes

• Definition: Multiple molecular forms of the same enzyme that catalyze the same reaction but differ in their physical, chemical, and kinetic properties. Encoded by different genes or result from different combinations of subunits.
• Example — LDH (Lactate Dehydrogenase): Tetramer of 2 subunit types: H (heart) and M (muscle)
  • LDH-1 (H₄): Heart, RBC — highest affinity for lactate (works aerobically)
  • LDH-2 (H₃M): Heart, RBC
  • LDH-3 (H₂M₂): Brain, lung
  • LDH-4 (HM₃): Muscle, liver
  • LDH-5 (M₄): Liver, skeletal muscle — works in anaerobic conditions
• Diagnostic use:
  • Myocardial infarction: LDH-1 > LDH-2 ("flipped pattern")
  • Liver disease: LDH-5 elevated
• Other examples: CK (CK-MB for heart), Alkaline phosphatase isoenzymes (bone, liver, placenta)

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b. Oxidative Phosphorylation

• Definition: The process by which ATP is synthesized using energy from the electron transport chain (ETC) driven by NADH and FADH₂ oxidation.
• Location: Inner mitochondrial membrane
• Components:
  • Complex I (NADH dehydrogenase): NADH → CoQ; pumps 4H⁺
  • Complex II (Succinate dehydrogenase): FADH₂ → CoQ; does NOT pump H⁺
  • Complex III (Cytochrome bc₁): CoQH₂ → Cyt c; pumps 4H⁺
  • Complex IV (Cytochrome c oxidase): Cyt c → O₂ → H₂O; pumps 2H⁺
  • Complex V (ATP synthase/F₀F₁-ATPase): H⁺ re-entry drives ATP synthesis
• Chemiosmotic theory (Mitchell): ETC pumps H⁺ from matrix to intermembrane space → electrochemical gradient (proton motive force, Δp) → H⁺ flows back through ATP synthase → ATP formed
• ATP yield: NADH → ~2.5 ATP; FADH₂ → ~1.5 ATP
• Inhibitors:
  • ETC: Rotenone (Complex I), Antimycin A (Complex III), Cyanide/CO (Complex IV)
  • ATP synthase: Oligomycin
  • Uncouplers (dissipate gradient without ATP): DNP (dinitrophenol), Thermogenin (brown fat)

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c. Enzyme Inhibitors

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d. Production and Fate of Pyruvate in the Body

1. Glycolysis (major source): Glucose → 2 Pyruvate (PEP → Pyruvate via pyruvate kinase)
2. Transamination of Alanine: Alanine + α-KG → Pyruvate + Glutamate (ALT reaction)
3. Serine deamination → Pyruvate
4. Malate → Pyruvate (malic enzyme, NADP⁺-dependent)

Pyruvate
├── Aerobic conditions:
│   ├── → Acetyl-CoA [Pyruvate dehydrogenase (PDH), irreversible]
│   │         → TCA cycle → CO₂ + H₂O + ATP
│   └── → OAA [Pyruvate carboxylase, biotin-dependent]
│             → Gluconeogenesis
│
├── Anaerobic conditions:
│   └── → Lactate [Lactate dehydrogenase (LDH)]
│             → regenerates NAD⁺ for continued glycolysis
│
└── Transamination:
    └── → Alanine [ALT] → glucose-alanine cycle

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e. Polyol Pathway in Uncontrolled Diabetes

Glucose →[Aldose reductase, NADPH]→ Sorbitol →[Sorbitol dehydrogenase, NAD⁺]→ Fructose

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f. Shuttle Systems (Cytosol → Mitochondria for NADH)

1. Malate-Aspartate Shuttle (Major, in heart/liver)

Cytosolic NADH → reduces OAA → Malate
Malate enters mitochondria → oxidized back to OAA → NADH (mitochondrial)
Net: 1 cytosolic NADH → 2.5 ATP (efficient)

2. Glycerol-3-Phosphate Shuttle (Brain, muscle)

Cytosolic NADH → reduces DHAP → Glycerol-3-phosphate
Enters mitochondria → oxidized by FAD-linked G3P dehydrogenase → FADH₂ (mitochondrial)
Net: 1 cytosolic NADH → 1.5 ATP (less efficient)

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g. Gluconeogenesis is NOT Simply Reversal of Glycolysis

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Q5. Short Explanations (Answer any 5)

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a. In Hypoxia, Concentration of 2,3-BPG Increases

• In hypoxia, RBCs shift to anaerobic glycolysis → more glucose-1,3-BPG is produced.
• Bisphosphoglycerate mutase (BPGM) converts 1,3-BPG → 2,3-BPG (Rapoport-Luebering shunt).
• 2,3-BPG binds to deoxyhemoglobin (in the central cavity of β-chains) → stabilizes the T (tense/deoxy) state → right shift of O₂-dissociation curve → ↓O₂ affinity → more O₂ released to tissues.
• This is a compensatory mechanism in hypoxia, high altitude, and chronic anemia.

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b. G-6-PD Deficiency Causes Hemolytic Anemia

• G6PD (Glucose-6-phosphate dehydrogenase) is the first and rate-limiting enzyme of the Pentose Phosphate Pathway (HMP shunt).
• It generates NADPH: G6P + NADP⁺ → 6-phosphogluconolactone + NADPH
• NADPH is essential to maintain reduced glutathione (GSH) via glutathione reductase.
• GSH protects RBCs from oxidative damage (neutralizes H₂O₂ via glutathione peroxidase).
• In G6PD deficiency: ↓NADPH → ↓GSH → oxidative stress → Heinz body formation (denatured hemoglobin) → RBC membrane damage → intravascular hemolysis.
• Triggered by: oxidant drugs (primaquine, dapsone, sulfonamides), fava beans, infections.
• X-linked recessive; common in malaria-endemic regions (provides some protection against P. falciparum).

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c. Lactate Production is Necessary During Anaerobic Glycolysis

• In anaerobic conditions, mitochondrial oxidative phosphorylation is halted → NAD⁺ is NOT regenerated by ETC.
• Glycolysis requires NAD⁺ (at the GAPDH step): G3P + NAD⁺ → 1,3-BPG + NADH.
• If NADH is not reoxidized, glycolysis stops.
• Lactate dehydrogenase (LDH): Pyruvate + NADH → Lactate + NAD⁺
• This regenerates NAD⁺ and allows glycolysis to continue, producing ATP (net 2 ATP/glucose).
• Lactate is transported to the liver → converted back to glucose (Cori cycle).
• In lactic acidosis (sepsis, tissue hypoxia, metformin toxicity): excess lactate accumulates → metabolic acidosis.

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d. Nucleotide Analogues Are Used as Anti-Cancer Agents

• Nucleotide analogues are structural mimics of purines or pyrimidines that interfere with DNA/RNA synthesis in rapidly dividing cancer cells.

• They are incorporated into DNA causing chain termination or inhibit enzymes of nucleotide synthesis → selectively target dividing cells.

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e. Allopurinol Is Used for Treatment of Gout

• Gout = hyperuricemia → monosodium urate crystal deposition in joints → acute inflammatory arthritis.
• Uric acid is the final product of purine catabolism (in humans, who lack uricase).
• Allopurinol (structural analogue of hypoxanthine):
  • Step 1: Metabolized by xanthine oxidase to oxypurinol (alloxanthine) — a suicide/mechanism-based inhibitor.
  • Step 2: Oxypurinol irreversibly inhibits xanthine oxidase.
  • Result: Hypoxanthine and xanthine accumulate (more soluble than uric acid) → excreted → ↓serum uric acid.
• Used in: chronic gout, uric acid nephrolithiasis, Lesch-Nyhan syndrome, tumor lysis syndrome (prophylaxis).
• Note: Allopurinol does NOT treat acute gout attacks (use colchicine/NSAIDs); it is for long-term urate lowering.

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f. ADA Deficiency Causes SCID

• ADA = Adenosine Deaminase — enzyme in purine catabolism.
• Reaction: Adenosine → Inosine (deamination); also deoxyadenosine → deoxyinosine
• In ADA deficiency: deoxyadenosine accumulates → converted to dATP by kinases.
• dATP accumulation is specifically toxic to lymphocytes:
  • Inhibits ribonucleotide reductase → ↓other dNTPs → impairs DNA synthesis.
  • Induces apoptosis of lymphocytes.
  • Both T and B lymphocytes are destroyed → SCID (Severe Combined Immunodeficiency).
• Clinical features: recurrent severe infections from birth, failure to thrive, absent thymic shadow.
• Treatment: Enzyme replacement therapy (PEG-ADA) or gene therapy (ADA-SCID was the first disease treated by gene therapy).

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Q6. MCQ

I. B DNA is:

• Double-stranded (two antiparallel complementary strands)
• Right-handed helix
• 10 base pairs per turn
• 3.4 Å rise per base pair
• 34 Å per complete turn
• Major and minor grooves present

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Summary Table of all correct answers to MCQ (for the full Q6 if more were visible):

• B DNA = Double stranded (option b)

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