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BIOCHEMISTRY PAPER-II - COMPLETE SOLUTIONS

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CASE SCENARIO 1

a. Most Likely Diagnosis (2 marks)

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b. Deficient Enzyme (2 marks)

• PAH normally converts phenylalanine → tyrosine
• It requires the cofactor tetrahydrobiopterin (BH4)
• A minority of PKU cases (~2%) are caused by deficiency of BH4 itself (dihydropteridine reductase deficiency); these are called "malignant" or "atypical" PKU

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c. Biochemical Basis of Elevated Blood Phenylalanine (3 marks)

1. Normal pathway blocked: PAH catalyzes the irreversible hydroxylation of phenylalanine to tyrosine. When PAH is absent/deficient, this pathway fails.
2. Accumulation: Phenylalanine cannot be converted to tyrosine, so it accumulates in blood, tissues, and CSF.
3. Alternative pathways activated: Excess phenylalanine is shunted into minor pathways:
   • Transamination → Phenylpyruvate (phenylketone; gives musty odor)
   • Reduction → Phenyllactate
   • Decarboxylation → Phenylethylamine These phenylketones are excreted in urine (phenylketonuria).
4. Neurotoxicity: High phenylalanine competes with other large neutral amino acids (e.g., tryptophan, tyrosine) for the blood-brain barrier transport system (LAT1 transporter), reducing synthesis of serotonin, dopamine, norepinephrine → seizures and developmental delay.

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d. Why Fair Skin and Light-Colored Hair? (3 marks)

• Tyrosine is the precursor for melanin (the pigment responsible for skin and hair color).
• Melanin synthesis pathway: Tyrosine → DOPA → Dopaquinone → Melanin, catalyzed by the enzyme tyrosinase.
• In PKU:
  1. PAH is deficient → tyrosine is not produced from phenylalanine
  2. Tyrosine becomes a conditionally essential amino acid (dietary intake alone is insufficient for melanin synthesis)
  3. Low tyrosine → reduced melanin synthesis → hypopigmentation of skin, hair, and eyes (fair/blonde appearance, blue eyes)
• Additionally, excess phenylalanine competitively inhibits tyrosinase, further reducing melanin production.

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e. Which Amino Acid Becomes Essential? (2 marks)

• Normally, tyrosine is a non-essential (dispensable) amino acid because it is synthesized from phenylalanine by PAH.
• In PKU, since PAH is deficient, tyrosine cannot be synthesized endogenously.
• Therefore, tyrosine must be supplied entirely through the diet → it becomes conditionally essential in PKU.

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f. Treatment (3 marks)

• Phenylalanine-restricted diet - low phenylalanine formula/diet started as early as possible after newborn screening (Guthrie test / tandem MS)
• Tyrosine supplementation - since tyrosine cannot be made endogenously
• Avoid high-phenylalanine foods: meat, fish, eggs, dairy, nuts, regular bread

• Sapropterin (BH4; Kuvan): Oral tetrahydrobiopterin 5-20 mg/kg/day - a cofactor that can reduce phenylalanine levels in BH4-responsive PKU patients (works only in patients with residual PAH activity, ~25-50% of classical PKU)
• Pegvaliase (Palynziq): A pegylated form of phenylalanine ammonia lyase that substitutes for PAH, metabolizing phenylalanine via an alternate route; used in adults with uncontrolled PKU

• Regular blood phenylalanine levels
• Dietary compliance monitoring
• Neurodevelopmental assessment

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LONG ESSAY QUESTION 1 (15 marks)

a. Definition of Translation + Structure and Functions of Ribosomes (3 marks)

• A site (Aminoacyl site): Binds incoming aminoacyl-tRNA; decoding site
• P site (Peptidyl site): Holds peptidyl-tRNA carrying the growing polypeptide chain
• E site (Exit site): Holds deacylated (empty) tRNA before it exits

1. Decoding: Small subunit reads the mRNA codon and ensures accurate codon-anticodon base pairing
2. Peptide bond formation: Large subunit (peptidyl transferase center, which is catalytic rRNA/ribozyme) catalyzes peptide bond formation
3. Translocation: Moves mRNA and tRNA through the ribosome
4. mRNA threading: Channels mRNA through the small subunit

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b. Structure of tRNA and Its Role in Protein Synthesis (3 marks)

1. Acceptor stem (3' end): All tRNAs end in the universal sequence 5'-CCA-3'. The amino acid is attached to the 3'-OH of the terminal adenosine. This is the site of aminoacylation.
2. D-arm (DHU arm): Contains the modified base dihydrouridine (DHU). Involved in tRNA recognition by aminoacyl-tRNA synthetases.
3. Anticodon arm: Contains the anticodon triplet (3 bases complementary to the mRNA codon). This is the most important functional region for decoding.
4. TψC arm: Contains the sequence thymidine-pseudouridine-cytidine (TψC). Important for ribosome binding.
5. Variable loop: Between the anticodon and TψC arms; size varies between different tRNA species.

1. Amino acid activation (aminoacylation): Aminoacyl-tRNA synthetase (one for each of 20 amino acids) charges tRNA with its specific amino acid in a two-step reaction requiring ATP:
   • Step 1: AA + ATP → Aminoacyl-AMP + PPi
   • Step 2: Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP
   • PPi is hydrolyzed → driving the reaction forward (high energy cost = 2 ATP equivalents)
2. Decoding: The anticodon of charged tRNA base-pairs with the mRNA codon at the A site (Wobble base pairing allows one tRNA to read multiple codons)
3. Adapter molecule: tRNA physically bridges the nucleic acid information in mRNA to the amino acid sequence of the protein - it is the "adapter" in Crick's adapter hypothesis

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c. Steps of Translation - Activation, Initiation, Elongation, Termination (6 marks)

Step 1: Activation of Amino Acids

• Each amino acid is activated by its specific aminoacyl-tRNA synthetase (also called amino acid activating enzyme)
• ATP → AMP + PPi; the energy is stored in the aminoacyl-tRNA (high-energy ester bond)
• These synthetases proofread to ensure correct amino acid attachment ("second genetic code")
• Cost: 2 ATP equivalents per amino acid

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Step 2: Initiation

1. 30S subunit + mRNA (Shine-Dalgarno sequence base-pairs with 16S rRNA)
2. Initiator tRNA: fMet-tRNA^fMet binds to the AUG start codon at P site
3. Requires initiation factors IF1, IF2 (GTP-dependent), IF3
4. 50S subunit joins → 70S initiation complex formed; GTP hydrolyzed; IFs released

1. eIF4E (cap-binding protein) recognizes the 5' m7G cap of mRNA
2. 40S small subunit + Met-tRNA^Met (initiator) + eIFs (eIF2·GTP) → 43S preinitiation complex
3. Complex scans mRNA 5'→3' until it finds AUG (Kozak sequence context)
4. 60S joins → 80S initiation complex; multiple eIFs required (eIF1, 1A, 2, 3, 4A, 4B, 4E, 4G, 5, 5B)

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Step 3: Elongation (repeating cycle)

• Elongation factor EF-Tu·GTP (prokaryote) / eEF1A·GTP (eukaryote) delivers aminoacyl-tRNA to the A site
• Correct codon-anticodon pairing → GTPase activation → GTP hydrolyzed → EF-Tu·GDP released
• Proofreading occurs (kinetic proofreading)
• Cost: 1 GTP

• The peptidyl transferase center (23S/28S rRNA acting as a ribozyme) catalyzes transfer of the growing peptide chain from peptidyl-tRNA (P site) to the aminoacyl-tRNA (A site)
• A new peptide bond forms; tRNA at P site becomes deacylated
• No energy input required - driven by thermodynamics

• Ribosome moves 3 nucleotides (one codon) in the 5'→3' direction
• Peptidyl-tRNA moves from A → P site
• Deacylated tRNA moves from P → E site (then exits)
• A site is now empty, ready for next aminoacyl-tRNA
• Requires EF-G·GTP (prokaryote) / eEF2·GTP (eukaryote); GTP hydrolyzed
• Cost: 1 GTP

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Step 4: Termination

• When a stop codon (UAA, UAG, or UGA) enters the A site, no normal tRNA can recognize it
• Release factors (RF) recognize stop codons:
  • Prokaryotes: RF1 (UAA, UAG), RF2 (UAA, UGA), RF3 (GTP-dependent, stimulates RF1/RF2)
  • Eukaryotes: eRF1 (all three stop codons), eRF3 (GTPase)
• RF stimulates peptidyl transferase to transfer the peptide to water (hydrolysis) → polypeptide released
• Ribosome recycling factor (RRF) + EF-G (prokaryotes) dissociate the 70S ribosome into subunits for reuse
• The completed polypeptide undergoes co-translational and post-translational modifications

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d. Inhibitors of Protein Synthesis (3 marks)

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SHORT ESSAY QUESTIONS (5 × 10 = 50 marks)

Attempt ANY 5

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1. Biologically Important Peptides

• Major intracellular antioxidant; protects cells from reactive oxygen species (H2O2, free radicals)
• Conjugates with drugs and toxins for detoxification (GSH-S-transferase)
• Maintains hemoglobin in reduced (Fe²⁺) state
• Deficiency → hemolytic anemia (e.g., G6PD deficiency causes GSH depletion)

• Released from posterior pituitary; stimulates uterine contractions during labor and milk ejection
• Has antidiuretic properties similar to vasopressin

• Released from posterior pituitary; acts on renal collecting ducts → water retention
• Raises blood pressure

• Most potent vasoconstrictor; stimulates aldosterone secretion from adrenal cortex
• Central to RAAS (Renin-Angiotensin-Aldosterone System)
• Formed from Angiotensin I by ACE (angiotensin-converting enzyme)

• Potent vasodilator; increases vascular permeability; mediates pain
• ACE inhibitors (antihypertensives) block bradykinin degradation → ACE inhibitor cough

• Endogenous opioid peptides; bind opioid receptors; mediate analgesia
• Met-enkephalin: Tyr-Gly-Gly-Phe-Met
• Leu-enkephalin: Tyr-Gly-Gly-Phe-Leu

• 51 amino acids (2 chains: A-chain 21 AA + B-chain 30 AA, linked by 2 disulfide bonds)
• Regulates blood glucose; promotes glucose uptake and glycogen synthesis

• Tripeptide (pyroGlu-His-Pro-NH2) from hypothalamus; stimulates TSH and prolactin release

• Present in muscle; acts as buffer (imidazole group of histidine); antioxidant

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2. Differential Diagnosis of Jaundice

A. Pre-hepatic (Hemolytic) Jaundice

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B. Hepatic (Hepatocellular) Jaundice

• Crigler-Najjar syndrome (Type I & II): UGT1A1 enzyme absent/reduced → unconjugated hyperbilirubinemia
• Gilbert's syndrome: Mild UGT1A1 reduction; mild unconjugated jaundice (benign)
• Dubin-Johnson syndrome: Defect in MRP2 (canalicular transport protein); conjugated hyperbilirubinemia; liver cells contain black pigment
• Rotor syndrome: Defect in OATP1B1/3 transporters; conjugated hyperbilirubinemia

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C. Post-hepatic (Obstructive/Cholestatic) Jaundice

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3. Role of 2,3-BPG in Oxygen Transport by Hemoglobin

• Formed from 1,3-bisphosphoglycerate (1,3-BPG) by BPG mutase (Rapoport-Luebering shunt - a bypass of glycolysis)
• This diverts 1,3-BPG away from ATP synthesis → RBCs can "waste" glycolytic energy to regulate O2 delivery

1. 2,3-BPG binds specifically to deoxyhemoglobin (T-state) in a central cavity formed by the β-chains (between the two β subunits)
2. Electrostatic interactions: 2,3-BPG (with 5 negative charges) binds to positively charged residues (Val-1, His-2, Lys-82, His-143) on each β-chain
3. Binding stabilizes the T (tense/deoxy) state of hemoglobin → reduces oxygen affinity
4. The O2-hemoglobin dissociation curve shifts to the right (Bohr effect-like shift)
5. This means Hb releases O2 more readily to tissues

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4. RNA Editing

A. C-to-U Editing (Cytidine deaminase mechanism)

• In the liver: Full-length ApoB-100 (4,536 AA) is translated from unedited mRNA
• In the intestine: An RNA-editing enzyme complex (APOBEC1 + ACF) deaminates a specific C → U at codon 2153
• This converts a CAA (glutamine codon) to UAA (stop codon)
• Result: Shorter ApoB-48 (2,152 AA) is produced
• ApoB-100 is used in LDL/VLDL; ApoB-48 is used in chylomicrons

B. A-to-I Editing (Adenosine deaminase mechanism)

• Adenosine deaminase acting on RNA (ADAR) enzymes deaminate A → I (inosine)
• Inosine is read as G by the translational machinery
• Example: Glutamate receptor (GluR-B subunit):
  • Editing changes CAG (Gln) → CIG (read as CGG = Arg) at the Q/R site
  • Unedited receptor: calcium-permeable; Edited receptor: calcium-impermeable
  • This editing is >99% efficient in the brain
• Example: Serotonin 2C receptor (5-HT2CR): Multiple editing sites alter receptor pharmacology and signaling

1. Generates protein diversity from a single gene (proteome expansion)
2. Regulates tissue-specific gene expression (liver vs. intestine ApoB)
3. Critical for neural function (GluR editing prevents excitotoxicity)
4. Dysregulation linked to neurological diseases, cancer, and viral defense
5. Innate immunity: ADAR editing of viral dsRNA can suppress interferon responses

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5. Biochemistry of AIDS

• Envelope: gp120 (outer) + gp41 (transmembrane) - form the envelope spike
• Core: p24 capsid protein, p7 nucleocapsid
• Enzymes: Reverse transcriptase (RT), Integrase, Protease (all encoded by pol gene)
• Genome: 2 copies of (+) ssRNA (~9.7 kb) encoding: gag, pol, env, and regulatory genes (tat, rev, nef, vif, vpr, vpu)

1. Attachment: gp120 binds CD4 receptor + co-receptor (CCR5 on macrophages; CXCR4 on T cells) on CD4+ T lymphocytes
2. Fusion: gp41 undergoes conformational change → fusion of viral and host membranes → RNA genome enters cytoplasm
3. Reverse transcription: RT (RNA-dependent DNA polymerase) converts ssRNA → dsDNA (also has RNase H activity to degrade RNA template):
   • ssRNA(+) → RNA:DNA hybrid → ssDNA → dsDNA
   • RT lacks 3'→5' proofreading → high mutation rate → rapid viral evolution, drug resistance
4. Integration: dsDNA + Integrase → covalent insertion into host chromosome as provirus (permanent; latent infection)
5. Transcription: Host RNA polymerase transcribes proviral DNA → viral mRNA; Tat protein (transactivator) dramatically enhances transcription
6. Translation: Host ribosomes translate viral mRNAs; Rev protein regulates export of unspliced/partially-spliced mRNAs from nucleus
7. Assembly & Budding: Gag and Gag-Pol polyproteins assemble at membrane
8. Maturation: HIV Protease cleaves polyproteins (Gag, Gag-Pol) into functional structural proteins and enzymes → mature, infectious virion

• NRTIs/NNRTIs - inhibit reverse transcriptase
• Integrase inhibitors (dolutegravir, raltegravir) - block integration
• Protease inhibitors - block viral maturation
• Entry inhibitors (maraviroc - CCR5 antagonist; enfuvirtide - fusion inhibitor)
• cART (combination antiretroviral therapy) - targets multiple steps simultaneously

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6. Structure and Classification of Immunoglobulins

• Light chains (L): ~214 amino acids; 2 types: κ (kappa) or λ (lambda); each has 1 variable (VL) + 1 constant (CL) domain
• Heavy chains (H): ~450-550 amino acids; each has 1 variable (VH) + 3 or 4 constant (CH1, CH2, CH3 ±CH4) domains; type determines Ig class
• Variable regions (Fab): The N-terminal portions of both H and L chains form the antigen-binding site (CDRs - complementarity-determining regions)
• Constant regions (Fc): The C-terminal portion of H chains; mediates effector functions (complement activation, FcR binding)
• Hinge region: Flexible proline/cysteine-rich region between CH1 and CH2; contains inter-chain disulfide bonds; gives flexibility

• Fab (Fragment antigen binding) = 1 VH + CH1 + entire light chain; antigen binding
• Fc (Fragment crystallizable) = 2 × (CH2 + CH3); effector functions
• Pepsin cleaves below hinge → F(ab')2 (2 antigen-binding arms linked) + pFc' (degraded)

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7. N2-Balance (Nitrogen Balance)

• N output = Urinary N (mainly urea ~80%) + Fecal N + Dermal N + Other losses

A. Positive Nitrogen Balance (N intake > N output)

• Body is retaining nitrogen → net protein synthesis exceeds protein breakdown
• Examples: Growing children, pregnancy, recovery from illness/surgery, athletes in training, anabolic steroid use, administration of growth hormone, insulin, testosterone

B. Zero/Neutral Nitrogen Balance (N intake = N output)

• Protein synthesis = protein breakdown
• Steady state in healthy adults
• Amino acid pool is maintained

C. Negative Nitrogen Balance (N intake < N output)

• Protein breakdown exceeds synthesis → net protein loss (muscle wasting/catabolism)
• Examples:
  • Protein deficiency (kwashiorkor), total starvation
  • Burns, trauma, major surgery, severe infections, febrile states
  • Cushing's syndrome (glucocorticoids promote proteolysis)
  • Immobilization, aging
  • Cancer cachexia, AIDS

• Protein turnover occurs continuously: ~200-300 g protein degraded and resynthesized daily
• Ubiquitin-proteasome system (UPS) is the main pathway for intracellular protein degradation
• The liver is the main site of amino acid catabolism and urea synthesis (urea cycle)
• Normal protein intake: ~0.8-1.0 g/kg/day (adults); increased in catabolic states, pregnancy

• Kwashiorkor = protein deficiency (adequate calories) → hypoalbuminemia → edema, fatty liver, skin/hair changes; N-balance severely negative
• Marasmus = total calorie deficiency → severe wasting of muscle and fat; adapted negative N-balance

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8. Post-Translational Modifications (PTMs)

• N-linked glycosylation: Carbohydrate attached to nitrogen of Asn (in Asn-X-Ser/Thr sequon); occurs in RER and Golgi; important for protein folding, stability, cell signaling
• O-linked glycosylation: Carbohydrate attached to oxygen of Ser or Thr; occurs in Golgi
• Example: IgG, erythropoietin, collagen

• By protein kinases (using ATP); removed by phosphatases
• Sites: Ser, Thr, Tyr (in eukaryotes)
• Major regulatory mechanism: enzyme activation/inactivation, signal transduction, cell cycle control

• Proline → Hydroxyproline; Lysine → Hydroxylysine (in collagen)
• Catalyzed by prolyl/lysyl hydroxylase (requires Vitamin C); essential for collagen stability (cross-linking)
• Deficiency of Vitamin C → Scurvy (defective collagen → bleeding, poor wound healing)

• Glutamate → γ-carboxyglutamate (Gla) by Vitamin K-dependent carboxylase
• Required for calcium-binding activity of clotting factors (II, VII, IX, X) and proteins C, S
• Warfarin inhibits Vitamin K epoxide reductase → inhibits carboxylation → anticoagulation

• Signal peptide removal (directs proteins to ER)
• Zymogen activation: trypsinogen → trypsin, pepsinogen → pepsin
• Proinsulin (86 AA) → Insulin (51 AA) + C-peptide by cleavage of 2 Arg-Arg sites

• N-terminal acetylation (by N-acetyltransferases): protects protein from degradation; improves membrane binding
• Histone acetylation: opens chromatin → promotes transcription (epigenetic regulation)

• Histone methylation regulates gene expression (epigenetic)
• Arg and Lys residues are methylated

• Attachment of ubiquitin (76 AA protein) to Lys residues; polyubiquitination → targets protein for proteasomal degradation
• Monoubiquitination → protein trafficking, DNA repair

• Between Cys residues; stabilizes tertiary and quaternary structure (e.g., insulin, immunoglobulins)

• Attach lipid groups to N-terminal Gly (myristoylation) or Cys residues (palmitoylation)
• Anchor proteins to plasma membrane (e.g., Src kinase, Ras)

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9. Salvage Pathway and Its Significance

• De novo synthesis: From small precursors (amino acids, CO2, ribose-5-phosphate); energy-intensive (~6 ATP per purine)
• Salvage pathway: Recycling of preformed purine/pyrimidine bases from nucleic acid degradation; much more energy-efficient

Purine Salvage Pathway

1. HGPRT (Hypoxanthine-Guanine Phosphoribosyl Transferase):
   • Hypoxanthine + PRPP → IMP (inosine monophosphate)
   • Guanine + PRPP → GMP (guanosine monophosphate)
   • PRPP = phosphoribosyl pyrophosphate (activated ribose-5-phosphate)
2. APRT (Adenine Phosphoribosyl Transferase):
   • Adenine + PRPP → AMP (adenosine monophosphate)

Clinical Significance

• X-linked recessive disorder; complete HGPRT deficiency
• Hypoxanthine and guanine cannot be salvaged → they are degraded to uric acid (via xanthine oxidase)
• Severe hyperuricemia → gout, nephrolithiasis, urate nephropathy
• Neurological: self-mutilation (biting lips, fingers), choreoathetosis, intellectual disability, spasticity
• Treatment: Allopurinol (xanthine oxidase inhibitor) reduces uric acid, but does NOT correct neurological symptoms (since neurological effects are from HGPRT deficiency itself, not uric acid)

• Less severe than Lesch-Nyhan; mainly hyperuricemia and gout

• Adenosine → Inosine step blocked
• Accumulation of deoxyadenosine and its phosphorylated derivatives (dATP)
• dATP is toxic to lymphocytes (especially T cells) → Severe Combined Immunodeficiency (SCID) - "bubble boy disease"

• 6-mercaptopurine (6-MP), 6-thioguanine: Converted by HGPRT to toxic nucleotide analogs → inhibit de novo purine synthesis and DNA synthesis → used in cancer chemotherapy (leukemia)
• Allopurinol: Substrate for HGPRT; converted to alloxanthine (oxypurinol) which inhibits xanthine oxidase

• Less clinically prominent; involves uridine kinase, thymidine kinase (TK)
• TK is used in HSV (herpes) treatment: acyclovir is phosphorylated by viral TK → active acyclovir triphosphate inhibits viral DNA polymerase (selectivity based on viral TK)

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10. Role of Physician in Health Care System

• Diagnosis, treatment, and management of diseases
• Ordering and interpreting investigations
• Prescribing medications appropriately (pharmacovigilance, antibiotic stewardship)
• Performing procedures and surgeries

• Vaccination, health screenings, early detection of disease
• Counseling patients about lifestyle modification (diet, exercise, smoking cessation)
• Implementing primary, secondary, and tertiary prevention
• Occupational health and environmental health assessments

• Health education to patients, families, and communities
• Promoting public awareness about communicable and non-communicable diseases
• Nutritional guidance and mental health promotion

• Assisting patients in recovery post-illness or injury
• Referral to physiotherapy, occupational therapy, counseling
• Chronic disease management (diabetes, hypertension, COPD)

• Contributing to clinical and translational research
• Applying evidence-based medicine to clinical decision-making
• Participating in clinical trials and post-marketing surveillance

• Hospital administration, policy development
• Health resource allocation
• Medicolegal responsibilities, medical documentation
• Maintaining confidentiality and ethical standards

• Addressing social determinants of health (poverty, education, sanitation)
• Participation in disease surveillance and outbreak management (epidemiology)
• Collaborating with public health authorities (WHO, national programs like RNTCP, NVBDCP)
• Medical ethics: beneficence, non-maleficence, autonomy, justice

• Teaching medical students, interns, and residents
• Continuous medical education (CME) for self-improvement

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