---

MBBS Biochemistry - Long Answer Questions 6 to 10

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QUESTION 6: Classify Amino Acids on the Basis of Nutritional and Metabolic Significance. Add a Note on Biologically Important Peptides.

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DEFINITION

        NH₂
        |
  R  -  Cα  -  COOH
        |
        H

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CLASSIFICATION I: NUTRITIONAL (DIETARY) SIGNIFICANCE

┌─────────────────────────────────────────────┐
│           AMINO ACIDS (Nutritional)          │
└──────────────────┬──────────────────────────┘
                   │
       ┌───────────┼───────────┐
       ▼           ▼           ▼
 ESSENTIAL    SEMI-ESSENTIAL  NON-ESSENTIAL
 (Indispens-  (Conditionally  (Dispensable)
  able)        Essential)

A. Essential Amino Acids (Cannot be synthesized; must be from diet)

B. Semi-Essential (Conditionally Essential)

• Arginine - essential during rapid growth, illness
• Histidine - essential in infants, uremia
• Cysteine - essential when methionine is deficient
• Tyrosine - essential when phenylalanine is deficient
• Glutamine - essential in critical illness, trauma

C. Non-Essential Amino Acids (Can be synthesized in the body)

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CLASSIFICATION II: METABOLIC SIGNIFICANCE

┌───────────────────────────────────────────────────┐
│        AMINO ACIDS (Metabolic Classification)      │
└──────────────────────┬────────────────────────────┘
                       │
           ┌───────────┼───────────┐
           ▼           ▼           ▼
     GLUCOGENIC    KETOGENIC    GLUCOGENIC +
                                KETOGENIC
                                (Both)

A. Glucogenic Amino Acids

• Degraded to pyruvate, oxaloacetate, α-ketoglutarate, succinyl-CoA, or fumarate
• Can be converted to glucose via gluconeogenesis
• Examples: Alanine, Glycine, Serine, Threonine, Aspartate, Asparagine, Glutamate, Glutamine, Arginine, Histidine, Valine, Methionine, Proline, Cysteine

B. Ketogenic Amino Acids

• Degraded to acetyl-CoA or acetoacetyl-CoA
• Cannot be converted to glucose; can form ketone bodies
• Only 2 purely ketogenic: Leucine, Lysine
  • Mnemonic: "Luckily Leucine & Lysine are Ketogenic"

C. Both Glucogenic AND Ketogenic

• Phenylalanine, Tyrosine, Tryptophan, Isoleucine, Threonine
  • Mnemonic: "PeTTy IT"

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METABOLIC FLOWCHART OF AMINO ACID FATES

  DIETARY PROTEIN
       │
       ▼ (Digestion by pepsin, trypsin, chymotrypsin)
  FREE AMINO ACIDS (in blood)
       │
  ┌────┼──────────────────────┐
  ▼    ▼                      ▼
PROTEIN  NITROGEN-          CARBON SKELETON
SYNTHESIS CONTAINING         │
          COMPOUNDS          ├──► Pyruvate ──► Glucose (gluconeogenesis)
          (heme,             │                (GLUCOGENIC)
          neurotransmitters, ├──► Acetyl-CoA ► Ketone bodies
          creatine,          │                (KETOGENIC)
          polyamines)        └──► TCA cycle ──► CO₂ + H₂O + Energy

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CLASSIFICATION III: BY SIDE CHAIN (R-group) CHARACTER

           AMINO ACIDS
               │
    ┌──────────┼──────────┐
    ▼          ▼          ▼
NONPOLAR   POLAR         ELECTRICALLY
(Hydro-    UNCHARGED     CHARGED
phobic)                  │
                     ┌───┴───┐
                     ▼       ▼
                  ACIDIC   BASIC
                  (Asp,    (Lys,
                   Glu)    Arg,
                           His)

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NOTE ON BIOLOGICALLY IMPORTANT PEPTIDES

  NH₂-AA₁-CO-NH-AA₂-CO-NH-AA₃-COOH
              │        │
           Peptide  Peptide
            Bond     Bond

Key Biologically Important Peptides:

Flowchart - Role of Glutathione:

  GSH (Reduced glutathione)
       │
       │ + H₂O₂ (toxic)
       ▼  [Glutathione peroxidase]
  GSSG (Oxidized glutathione) + H₂O
       │
       │ [Glutathione reductase + NADPH]
       ▼
  GSH (regenerated)

  ─────────────────────────────
  NADPH comes from G6P via HMP shunt
  Deficiency of G6PD → ↓NADPH → ↓GSH → Hemolytic anemia

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QUESTION 7: Define Proteins. Write in Detail About the Structural Organization of Proteins. Add a Note on Disorders Associated with Misfolded Proteins.

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DEFINITION

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STRUCTURAL ORGANIZATION OF PROTEINS

        PRIMARY STRUCTURE
              │
              ▼
        SECONDARY STRUCTURE
              │
              ▼
        TERTIARY STRUCTURE
              │
              ▼
        QUATERNARY STRUCTURE
         (only in multi-subunit proteins)

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LEVEL 1: PRIMARY STRUCTURE

• The linear sequence of amino acids in the polypeptide chain
• Amino acids are linked by covalent peptide bonds (-CO-NH-)
• Determines all higher levels of structure
• Alterations (point mutations) cause diseases (e.g., sickle cell anemia)

  H₂N - AA₁ - AA₂ - AA₃ - AA₄ - ... - AAn - COOH
               │         │
           Peptide    Peptide
            bond       bond

---

LEVEL 2: SECONDARY STRUCTURE

• Local folding of the polypeptide backbone stabilized by hydrogen bonds between backbone NH and C=O groups
• Two main types:

  SECONDARY STRUCTURE
         │
    ┌────┴────┐
    ▼         ▼
  α-HELIX   β-PLEATED
            SHEET
    │           │
  Right-hand  Parallel or
  coiled helix anti-parallel
    │
  3.6 AA/turn
  rise = 1.5 Å/residue
  pitch = 5.4 Å

• Right-handed helix
• H-bonds between C=O of residue n and N-H of residue n+4
• Examples: Hemoglobin, myoglobin, keratin

• Extended strands connected by H-bonds perpendicular to the chain
• Parallel: both chains run N→C in same direction
• Anti-parallel: chains run in opposite directions
• Example: Silk fibroin, immunoglobulins

• β-turns / β-bends: Sharp 180° turns; 4 residues; stabilized by H-bonds
• Collagen triple helix: Three left-handed helices wound together (Gly-X-Y repeats)

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LEVEL 3: TERTIARY STRUCTURE

• Overall 3D folding of the entire polypeptide chain
• Stabilized by multiple non-covalent and covalent forces:

  FORCES STABILIZING TERTIARY STRUCTURE
             │
    ┌─────────┼──────────┬──────────┐
    ▼         ▼          ▼          ▼
 Hydrogen  Hydrophobic  Ionic    Disulfide
  bonds    interactions  bonds     bonds
 (weak)    (strongest   (salt    (covalent -
            driving     bridges)  S-S- bonds)
            force)

• Hydrophobic core: Nonpolar side chains buried away from water
• Hydrophilic surface: Polar/charged groups face the aqueous environment
• Domain: Compact globular region with specific function (e.g., DNA-binding domain, active site)

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LEVEL 4: QUATERNARY STRUCTURE

• Association of two or more polypeptide subunits (monomers) into a larger functional complex
• Subunits held together by same non-covalent forces as tertiary structure
• Each subunit has its own tertiary structure

  Example: HEMOGLOBIN
    2α subunits + 2β subunits
           │
           ▼
    α₁β₁ + α₂β₂ tetramer
    (MW ~64,500 Da)
    Each subunit carries 1 heme

• LDH (Lactate Dehydrogenase): tetramer (4 subunits)
• Immunoglobulin IgG: 4 chains (2H + 2L)
• Collagen: Triple helix

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SUMMARY DIAGRAM: LEVELS OF PROTEIN STRUCTURE

PRIMARY     ──► Linear sequence: -AA1-AA2-AA3-AA4-
                                    Peptide bonds

SECONDARY   ──► Local folding:
                  α-helix (H-bonds in backbone, same chain)
                  β-sheet (H-bonds between adjacent chains)

TERTIARY    ──► 3D globular shape:
                  H-bonds + Hydrophobic + Ionic + S-S bonds

QUATERNARY  ──► Multi-subunit complex:
                  e.g., Hemoglobin = 2α + 2β subunits

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NOTE: DISORDERS ASSOCIATED WITH MISFOLDED PROTEINS (PROTEINOPATHIES)

  NORMAL PROTEIN SYNTHESIS
        │
        ▼
  Polypeptide chain
        │
        ▼ [Chaperones assist]
  Correctly folded protein ──► Function
        │
     FAILURE
        │
        ▼
  Misfolded protein
        │
   ┌────┴────┐
   ▼         ▼
DEGRADED  AGGREGATION
(Proteasome)  │
              ▼
        DISEASE

Key Misfolded Protein Disorders:

Prion Disease - Special Mechanism:

  Normal PrPc (α-helix rich)
       │
       │ [Misfolding triggered by
       │  abnormal PrPsc contact]
       ▼
  PrPsc (β-sheet rich, protease-resistant)
       │
       ▼ [Propagates exponentially]
  Amyloid plaques in brain
       │
       ▼
  Spongiform neurodegeneration
  (CJD, Fatal Familial Insomnia, Kuru)

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QUESTION 8: Describe the Functions of Plasma Proteins. Add a Note on Clinical Significance of Acute Phase Proteins.

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PLASMA PROTEINS - OVERVIEW

  PLASMA PROTEINS
         │
    ┌────┴─────┬──────────┬──────────┐
    ▼          ▼          ▼          ▼
  ALBUMIN  GLOBULINS  FIBRINOGEN  OTHERS
  (3.5-5  (α1, α2,    (0.2-0.4   (Complement,
  g/dL)   β, γ)       g/dL)       enzymes,
                                   hormones)

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FUNCTIONS OF PLASMA PROTEINS

1. Albumin (Major plasma protein: 50-60% of total)

  ALBUMIN FUNCTIONS
       │
    ┌──┴──────────────────────────┐
    ▼                             ▼
COLLOIDAL OSMOTIC              TRANSPORT
PRESSURE (COP)                 FUNCTION
(75-80% of total COP)              │
    │                       ┌──────┼───────┐
Maintains fluid             ▼      ▼       ▼
in vessels               Bilirubin  Fatty  Drugs
(prevents edema)         (unconjug) acids  (warfarin,
                                          penicillin)
                         Hormones   Ca²⁺  Tryptophan

2. Globulins

3. Fibrinogen

• Precursor of fibrin (clot formation)
• Converted by thrombin: Fibrinogen → Fibrin
• Also an acute phase protein

4. Buffer Function

• Plasma proteins act as buffers (carry H⁺ at histidine residues)
• Responsible for ~15% of blood buffering capacity

5. Viscosity of Blood

• Proteins (especially fibrinogen) contribute to blood viscosity

6. Enzymatic Functions

• Pseudocholinesterase: Hydrolyzes succinylcholine
• Lipoprotein lipase, LCAT (lecithin-cholesterol acyl transferase)

7. Nutritional Reserve

• Amino acids from plasma proteins can be mobilized during starvation

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FLOWCHART: CAUSES OF HYPOALBUMINEMIA

  LOW ALBUMIN (< 3.5 g/dL)
         │
    ┌────┴────────┬──────────────┐
    ▼             ▼              ▼
  ↓ SYNTHESIS  ↑ LOSS        ↓ INTAKE
  (Liver       │              (Malnutrition,
  disease:     ├─ Nephrotic    Kwashiorkor)
  cirrhosis,   │  syndrome
  hepatitis)   ├─ Protein-losing
               │  enteropathy
               └─ Burns

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NOTE: ACUTE PHASE PROTEINS (APPs)

• IL-1, IL-6, TNF-α (cytokines released from macrophages at inflammation site)

Flowchart of Acute Phase Response:

  TISSUE INJURY / INFECTION / INFLAMMATION
               │
               ▼
    Macrophage/Monocyte activation
               │
               ▼
     Release of CYTOKINES:
     IL-1, IL-6, TNF-α
               │
               ▼
          LIVER
               │
    ┌──────────┼───────────────┐
    ▼          ▼               ▼
  ↑ POSITIVE   ↑ FIBRINOGEN    ↓ NEGATIVE
   APPs                         APPs

POSITIVE ACUTE PHASE PROTEINS (Increase):

NEGATIVE ACUTE PHASE PROTEINS (Decrease):

Clinical Significance of CRP:

  CRP < 1 mg/L     → Normal
  CRP 1-10 mg/L    → Mild inflammation, viral infection
  CRP 10-100 mg/L  → Bacterial infection, RA, IBD
  CRP > 100 mg/L   → Severe bacterial infection, sepsis
  CRP > 200 mg/L   → Serious bacterial infection

• CRP 1-3 mg/L → Intermediate CV risk
• CRP > 3 mg/L → High CV risk (cardiovascular risk marker)

• Autosomal recessive
• ↓ Inhibition of neutrophil elastase → emphysema (lung destruction)
• Misfolded protein accumulates in liver → cirrhosis

---

QUESTION 9: Describe the Structure of Immunoglobulin. Classify Immunoglobulins Along with Their Functions. Explain Paraproteinemias in Brief.

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DEFINITION

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BASIC STRUCTURE OF IMMUNOGLOBULIN (IgG as prototype)

         Fab region (antigen binding)
         │           │
    ┌────┴───┐   ┌───┴────┐
    │  VH  VL│   │VH  VL  │
    │  CH1 CL│   │CH1  CL │
    └────┬───┘   └───┬────┘
         │    HINGE  │
         └─────┬─────┘
               │ ← Fc region (effector function)
           ┌───┴───┐
           │  CH2  │
           │  CH3  │
           └───────┘

Components:

• 2 Heavy chains (H): ~50,000 Da each; define the class (IgG, IgA, IgM, IgD, IgE)
• 2 Light chains (L): ~25,000 Da each; two types: κ (kappa) and λ (lambda)
• Chains joined by disulfide bonds (-S-S-)

• Variable region (V): N-terminal; contains antigen-binding site (CDRs)
• Constant region (C): C-terminal; mediates effector functions

  IgG digested by PAPAIN → 2 Fab + 1 Fc

  IgG digested by PEPSIN → 1 F(ab')₂ + pFc' (degraded)

• Between CH1 and CH2
• Gives flexibility for antigen binding
• Rich in proline and cysteine (disulfide bonds)
• Absent in IgM and IgE

---

CLASSIFICATION OF IMMUNOGLOBULINS

  IMMUNOGLOBULINS
         │
    ┌────┼─────┬─────┬─────┐
    ▼    ▼     ▼     ▼     ▼
  IgG  IgA  IgM  IgD  IgE

Comparison Table:

Individual Functions:

• Most abundant; main antibody of secondary immune response
• Only immunoglobulin to cross the placenta (passive immunity to newborn)
• Opsonization, ADCC, complement activation
• Neutralizes toxins, viruses

• Present in secretions (saliva, tears, milk, colostrum, GI secretions) as secretory IgA (sIgA)
• First line of defense at mucosal surfaces
• Dimer joined by J chain + secretory component
• Protects against GI and respiratory infections

• Pentamer (5 monomers joined by J chain)
• First antibody produced in primary immune response
• Most potent activator of complement (classical pathway)
• Blood group antibodies (anti-A, anti-B) are IgM
• ABO incompatibility reactions

• Monomer on B-cell surface (B-cell receptor)
• Function: B-cell activation, differentiation signal
• Clinical significance: minimal

• Lowest concentration in serum
• Binds to mast cells and basophils via Fc receptor (FcεRI)
• Mediates Type I hypersensitivity (anaphylaxis, allergic asthma, urticaria)
• Anti-parasite immunity (eosinophil activation)

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FLOWCHART: ANTIBODY-MEDIATED IMMUNE RESPONSE

  ANTIGEN ENTRY
        │
        ▼
  Antigen processing by APC
        │
        ▼
  CD4+ T-helper cell activation
        │
        ▼
  B-cell activation + proliferation
        │
   ┌────┴────┐
   ▼         ▼
PLASMA    MEMORY
 CELLS    B-CELLS
   │
   ▼
ANTIBODY PRODUCTION
   │
   ├──► PRIMARY RESPONSE: IgM first, then IgG
   └──► SECONDARY RESPONSE: Rapid, ↑↑ IgG (class switching)

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NOTE: PARAPROTEINEMIAS

Definition:

  PARAPROTEINEMIA
        │
   ┌────┴────────────────┐
   ▼                     ▼
BENIGN                MALIGNANT
   │                     │
Monoclonal            ┌──┴─────────────────┐
Gammopathy of         ▼                    ▼
Undetermined       MULTIPLE           WALDENSTROM'S
Significance       MYELOMA            MACROGLOBULINEMIA
(MGUS)            (IgG >IgA)         (IgM)

Multiple Myeloma:

• Malignant proliferation of a single clone of plasma cells in bone marrow
• Produces M-spike on serum protein electrophoresis (usually IgG or IgA)
• Bence Jones proteinuria: Free light chains (κ or λ) in urine
• Features: Bone pain, lytic lesions, anemia, hypercalcemia, renal failure, recurrent infections
• Diagnosis: CRAB criteria - Hypercalcemia, Renal failure, Anemia, Bone lesions

Electrophoresis Pattern:

  NORMAL:        ─────/─────\─────────────────────
  Albumin ↑↑     γ region normal
  
  MULTIPLE       ─────/─────\──────────/\──────────
  MYELOMA:       Normal      M-spike (monoclonal band)
                             in γ (or β) region
  
  POLYCLONAL     ─────/─────\─────────/──────/────
  (Inflammation):            ↑↑ broad γ zone

Waldenstrom's Macroglobulinemia:

• Malignant B-cell lymphoma secreting IgM (pentamer, MW 900 kDa)
• Causes hyperviscosity syndrome: headache, visual disturbances, bleeding
• Treatment: Plasmapheresis

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QUESTION 10: Define Lipids. Classify with Suitable Examples. Add a Note on Functions and Clinical Significance of Phospholipids.

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DEFINITION OF LIPIDS

• Insoluble in water (hydrophobic)
• Soluble in organic solvents (chloroform, ether, benzene)
• Contain C, H, O (some also have N, P, S)
• Chemically, they are esters or potential esters of fatty acids

---

CLASSIFICATION OF LIPIDS

  LIPIDS
    │
    ├─ 1. SIMPLE LIPIDS (esters of fatty acids + alcohol)
    │       ├─ Fats (Triacylglycerols / Triglycerides)
    │       └─ Waxes (fatty acid + long chain alcohol)
    │
    ├─ 2. COMPOUND LIPIDS (simple lipid + additional group)
    │       ├─ Phospholipids
    │       │     ├─ Glycerophospholipids (glycerol backbone)
    │       │     │     ├─ Phosphatidylcholine (lecithin)
    │       │     │     ├─ Phosphatidylethanolamine (cephalin)
    │       │     │     ├─ Phosphatidylserine
    │       │     │     ├─ Phosphatidylinositol
    │       │     │     └─ Cardiolipin (diphosphatidylglycerol)
    │       │     └─ Sphingomyelin (sphingosine backbone)
    │       │
    │       ├─ Glycolipids
    │       │     ├─ Cerebrosides (galactose/glucose + ceramide)
    │       │     ├─ Gangliosides (+ sialic acid)
    │       │     └─ Sulfatides
    │       │
    │       └─ Lipoproteins (lipid + protein)
    │             ├─ Chylomicron
    │             ├─ VLDL
    │             ├─ IDL
    │             ├─ LDL
    │             └─ HDL
    │
    └─ 3. DERIVED LIPIDS (hydrolysis products)
            ├─ Fatty acids (saturated / unsaturated)
            ├─ Glycerol
            ├─ Sterols (cholesterol, ergosterol)
            ├─ Bile acids
            ├─ Steroid hormones
            ├─ Fat-soluble vitamins (A, D, E, K)
            └─ Ketone bodies

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FATTY ACIDS

  FATTY ACIDS
      │
   ┌──┴──────────────┐
   ▼                 ▼
SATURATED         UNSATURATED
(No double bond)  (Has double bonds)
   │                 │
Examples:        ┌───┴───┐
Palmitic (C16)   ▼       ▼
Stearic (C18)  MONO-   POLY-
               UNSATU-  UNSATU-
               RATED    RATED
               │        │
              Oleic    Linoleic (ω-6)
              (C18:1)  Linolenic (ω-3)
                       Arachidonic (ω-6)
                       EPA, DHA (ω-3)

• Linoleic acid (ω-6, C18:2) - Precursor of arachidonic acid
• α-Linolenic acid (ω-3, C18:3) - Precursor of EPA and DHA
• Deficiency: Dermatitis, poor wound healing, growth retardation, increased susceptibility to infection

---

TRIACYLGLYCEROLS (Triglycerides)

  Glycerol + 3 Fatty Acids
       │
       ▼
  Triacylglycerol (TAG)
  
    CH₂-O-CO-R₁
    │
    CH -O-CO-R₂     (R = fatty acid chain)
    │
    CH₂-O-CO-R₃

• Storage form of energy (9 kcal/g)
• Stored in adipose tissue
• Mobilized as free fatty acids during starvation

---

STRUCTURE OF PHOSPHOLIPIDS

        PHOSPHOLIPID STRUCTURE
  
   ─────────────────────────────────────
   POLAR HEAD (hydrophilic)
     │
   Phosphate ── Base (choline/ethanolamine/serine/inositol)
     │
   Glycerol
     │
   ├─ sn-1: Saturated fatty acid
   └─ sn-2: Unsaturated fatty acid
  
   NON-POLAR TAIL (hydrophobic)
   ─────────────────────────────────────

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FUNCTIONS OF PHOSPHOLIPIDS

1. Cell Membrane Structure

  ┌──────────────────────────────────────┐
  │ ○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○  │ Polar heads
  │ ||||||||||||||||||||||||||||||||||||  │ 
  │ ||||||||||||||||||||||||||||||||||||  │ Hydrophobic core
  │ ○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○○  │ Polar heads
  └──────────────────────────────────────┘
  Phospholipid bilayer forms the structural basis of ALL membranes

2. Lung Surfactant

• Dipalmitoylphosphatidylcholine (DPPC / Lecithin) - main component of pulmonary surfactant
• Reduces surface tension in alveoli; prevents alveolar collapse
• Lecithin:Sphingomyelin (L:S) ratio >2 indicates fetal lung maturity
• L:S < 2 → Risk of Neonatal Respiratory Distress Syndrome (NRDS)

3. Signal Transduction

  Phosphatidylinositol-4,5-bisphosphate (PIP₂)
       │
       │ [Phospholipase C activated by G-protein]
       ▼
  IP₃ (Inositol triphosphate)  +  DAG (Diacylglycerol)
       │                              │
       ▼                              ▼
  ↑ Intracellular Ca²⁺           Protein Kinase C activation
  (ER release)                   (Cell growth, proliferation)

4. Precursor for Eicosanoids

• Phosphatidylinositol and Phosphatidylcholine release arachidonic acid via phospholipase A₂
• Arachidonic acid → Prostaglandins, Thromboxanes, Leukotrienes (eicosanoids)
• These mediate inflammation, fever, pain, platelet aggregation

5. Blood Coagulation

• Phosphatidylserine on platelet surface (flips from inner to outer leaflet upon activation)
• Provides surface for assembly of coagulation complexes (tenase, prothrombinase)

6. Bile Formation and Fat Absorption

• Lecithin (phosphatidylcholine) in bile emulsifies fats for digestion
• Forms mixed micelles with bile salts and cholesterol

7. Lipoprotein Structure

• Phospholipids form the outer coat of lipoproteins (VLDL, LDL, HDL)
• Enable hydrophobic lipids to be transported in aqueous plasma

---

CLINICAL SIGNIFICANCE OF PHOSPHOLIPIDS

  PHOSPHOLIPID CLINICAL SIGNIFICANCE
               │
    ┌──────────┼──────────────┬────────────────┐
    ▼          ▼              ▼                ▼
   NRDS    ANTIPHOSPHO-   LECITHIN-      GAUCHER'S/
           LIPID          SPHINGOMYELIN  NIEMANN-PICK
           SYNDROME       RATIO          DISEASE

Key Clinical Conditions:

Antiphospholipid Syndrome (APS):

  Autoimmune condition
       │
       ▼
  Antibodies against phospholipids/
  phospholipid-binding proteins
  (anticardiolipin, anti-β2GPI, lupus anticoagulant)
       │
       ▼
  ┌────┴─────────────────┐
  ▼                      ▼
ARTERIAL THROMBOSIS   VENOUS THROMBOSIS
(Stroke, MI)          (DVT, PE)
       │
       ▼
RECURRENT PREGNANCY LOSS
(fetal loss, placental thrombosis)

• Associated with SLE
• Paradox: Lupus anticoagulant prolongs aPTT in vitro, but CAUSES thrombosis in vivo

---

CHOLESTEROL (Important Derived Lipid)

  CHOLESTEROL
       │
    ┌──┴────────────────────────────┐
    ▼                               ▼
  ENDOGENOUS                    DIETARY
  (Liver synthesizes             (Animal products)
  ~800-1000 mg/day)
       │
    ┌──┴────────────────────────────────────────┐
    ▼          ▼          ▼          ▼          ▼
  Cell      Bile      Steroid     Vitamin D  Myelin
  Membrane  Acids     Hormones    synthesis  sheath
  (fluidity) (digestion) (cortisol,
                        sex hormones)

---

SUMMARY TABLE - LIPID CLASSES

---

---

Exam Tips:

• Q6: Always include the mnemonic PVT TIM HaLL for essential amino acids and Luckily Leucine & Lysine are Ketogenic. Glutathione = Glu-Cys-Gly tripeptide is a frequent SAQ.
• Q7: Draw all 4 levels clearly. Sickle cell anemia and prion diseases are high-yield examples.
• Q8: CRP is the most sensitive and specific acute phase reactant. Negative APPs (albumin, transferrin) are commonly asked.
• Q9: IgG crosses placenta, IgA is in secretions, IgM is the pentamer first antibody. M-spike + Bence Jones protein = Multiple Myeloma.
• Q10: L:S ratio >2 = lung maturity. Antiphospholipid syndrome paradox (prolong aPTT but cause thrombosis) is a classic MCQ trap.

---

MBBS Biochemistry - Long Answer Questions 11 to 17

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QUESTION 11: Define Nucleotide and Nucleoside. Outline and Describe the Structural Features of DNA Double Helix. Add a Note on Clinical Applications of Synthetic Nucleotide Analogs.

---

DEFINITIONS

Nucleoside

• The base is attached to sugar by a N-glycosidic bond (at N-1 of pyrimidine, N-9 of purine)

Nucleotide

• Also called mononucleotide
• Nucleotide = Nucleoside + Phosphoric acid

  NUCLEOSIDE:
  Base ──── Sugar
  (N-glycosidic bond)

  NUCLEOTIDE:
  Base ──── Sugar ──── Phosphate
                       (phosphoester bond)

Difference Table:

---

NITROGENOUS BASES

  NITROGENOUS BASES
         │
    ┌────┴────┐
    ▼         ▼
 PURINES    PYRIMIDINES
 (2 rings)  (1 ring)
    │            │
  Adenine    Cytosine
  Guanine    Thymine (DNA only)
             Uracil (RNA only)

Mnemonic:
PURines = PURe As Gold (Adenine + Guanine)
CUT the PY-rimidines (Cytosine, Uracil, Thymine)

• DNA: Contains 2-deoxyribose (missing OH at C-2')
• RNA: Contains ribose (has OH at C-2')

---

WATSON-CRICK DNA DOUBLE HELIX - STRUCTURAL FEATURES

General Architecture:

  5'────────────────────────────3'
  │  P─S─[A]═════[T]─S─P      │
  │  P─S─[G]≡≡≡≡≡[C]─S─P      │
  │  P─S─[T]═════[A]─S─P      │
  │  P─S─[C]≡≡≡≡≡[G]─S─P      │
  3'────────────────────────────5'
  
  S = Deoxyribose sugar
  P = Phosphate
  = = 2 H-bonds (A-T)
  ≡ = 3 H-bonds (G-C)
  (strands are ANTIPARALLEL)

Key Structural Features:

---

COMPLEMENTARY BASE PAIRING & CHARGAFF'S RULES

  CHARGAFF'S RULES:
  
  A + G = C + T  (purines = pyrimidines)
  A = T
  G = C
  (A + T) / (G + C) = species-specific constant

---

GROOVES OF DNA

  DNA DOUBLE HELIX - Cross-section view:
  
       ┌──────────┐
       │   MAJOR  │ ← Transcription factors, repressors bind here
       │  GROOVE  │   Wide, accessible
       └──────────┘
            │
        bases stacked
            │
       ┌──────────┐
       │   MINOR  │ ← Some drugs (e.g., netropsin, berenil) bind here
       │  GROOVE  │   Narrow
       └──────────┘

---

DNA CONFORMATIONS - COMPARISON TABLE (for Q12 as well)

  DNA CONFORMATIONS
        │
   ┌────┼────┐
   ▼    ▼    ▼
  B-DNA A-DNA Z-DNA

---

FORCES STABILIZING DNA DOUBLE HELIX

  DNA STABILITY
       │
    ┌──┴──────────────────────┐
    ▼                         ▼
HYDROGEN BONDS             BASE STACKING
(between base pairs)       (hydrophobic
A=T: 2 H-bonds             interactions
G≡C: 3 H-bonds             between adjacent
                            base pairs)
                            ↑ Major stabilizing force

• Ionic interactions (phosphate + metal ions/histones)
• Van der Waals forces

---

NOTE: CLINICAL APPLICATIONS OF SYNTHETIC NUCLEOTIDE ANALOGS

  SYNTHETIC NUCLEOTIDE ANALOG
             │
        ┌────┴────┐
        ▼         ▼
   ANTICANCER   ANTIVIRAL
   AGENTS       AGENTS

A. Anticancer Nucleotide Analogs:

B. Antiviral Nucleotide Analogs:

Mechanism Flowchart (AZT):

  HIV enters cell → Reverse transcriptase copies RNA→DNA
                                │
                     AZT (triphosphate form)
                     competes with dTTP
                                │
                     Incorporated into viral DNA
                                │
                     No 3'-OH group → CHAIN TERMINATION
                                │
                     Viral DNA synthesis stops
                                │
                     ↓ HIV replication

---

QUESTION 12: Compare the Structural Features of Different Conformations of DNA Double Helix. Add a Note on Functions of Biologically Important Nucleotides.

---

DNA CONFORMATIONS (Detailed - see table in Q11 above for comparison)

B-DNA:

• Most common physiological form
• Regular right-handed helix, 10 bp/turn
• Watson-Crick model (1953)

A-DNA:

• Occurs under dehydrating conditions
• Also the form of RNA:DNA hybrid duplexes and double-stranded RNA
• Shorter, wider helix; minor groove wider

Z-DNA:

• Left-handed helix (zig-zag backbone, hence "Z")
• Occurs in GC-rich sequences under high salt
• Possible role in transcriptional regulation (Z-DNA binding proteins)
• Backbone follows a zig-zag path

  COMPARISON DIAGRAM:

  B-DNA         A-DNA         Z-DNA
    /─\            /─\         \─/
   │   │          │   │        │ │
    \─/            \─/         / \
    /─\            /─\        │   │
    ↑              ↑           ↑
  Right-         Right-      Left-
  handed         handed      handed
  10 bp/turn     11 bp/turn  12 bp/turn
  Most common    Low humidity  GC-rich

---

FUNCTIONS OF BIOLOGICALLY IMPORTANT NUCLEOTIDES

1. Building Blocks of Nucleic Acids

  NTPs (ATP, GTP, CTP, UTP) ──► RNA synthesis (transcription)
  dNTPs (dATP, dGTP, dCTP, dTTP) ──► DNA synthesis (replication)

2. Energy Currency - ATP

  GLUCOSE
     │ (Glycolysis, TCA, Oxidative phosphorylation)
     ▼
  ATP (Adenosine Triphosphate)
     │
  ┌──┴──────────────────────────────────┐
  ▼    ▼          ▼          ▼          ▼
Muscle Biosynthesis Active   Signal    Heat
  con- reactions  transport  transduc-
traction          (Na+/K+    tion
                  ATPase)

3. Coenzymes/Cofactors

4. Second Messengers

  HORMONE binds receptor
        │
        ▼
  Adenylyl cyclase activated
        │
        ▼
  ATP ──► cAMP (cyclic 3',5'-AMP)
        │
        ▼
  Protein Kinase A (PKA) activation
        │
        ▼
  PHOSPHORYLATION of target proteins
        │
        ▼
  CELLULAR RESPONSE

5. Allosteric Regulators

  AMP ──► activates phosphofructokinase-1 (PFK-1) [↑ glycolysis when energy is low]
  ATP ──► inhibits PFK-1 [↓ glycolysis when energy is adequate]
  ADP/AMP ratio ──► signals metabolic state of cell

6. Activated Intermediates in Biosynthesis

7. Signal Transduction (GTP)

• GTP bound to G-proteins (Gs, Gi) mediates receptor signaling
• GTP powers tubulin polymerization (mitotic spindle)
• GTP drives protein synthesis (elongation factors EF-Tu, EF-G use GTP)

---

QUESTION 13: Describe the Types, Structure and Functions of RNA.

---

DEFINITION

---

TYPES OF RNA - OVERVIEW FLOWCHART

  RNA (Ribonucleic Acid)
         │
    ┌────┴──────────────────────────────────┐
    ▼         ▼          ▼          ▼       ▼
  mRNA      tRNA       rRNA      snRNA   miRNA/siRNA
(Messenger)(Transfer)(Ribosomal)(Small   (Regulatory)
           (Adaptor)            nuclear)

---

1. mRNA (Messenger RNA)

  5'CAP──────────────────────────3'PolyA TAIL
  │    │                     │
  5'UTR  CODING SEQUENCE      3'UTR
         (AUG → STOP codon)
         Start codon

• 5' Cap (7-methylguanosine cap): Protection from exonucleases; ribosome recognition
• 5' UTR (untranslated region): Ribosome binding site
• Coding sequence: Contains codons; start codon AUG (Met), stop codons UAA, UAG, UGA
• 3' UTR: Regulation of stability and translation
• 3' Poly-A tail (~200 A residues): Stability, nuclear export, translation initiation

• Carries genetic code from nucleus to cytoplasm
• Template for protein synthesis (translation)
• Most unstable RNA (half-life minutes to hours in prokaryotes)

---

2. tRNA (Transfer RNA)

       3'  A
           C
           C
  5' ────── │ ──── Acceptor stem (Amino acid attachment site)
           │
      ┌────┘
      │   T-ψ-C loop (binds large rRNA)
      │
      │   Variable loop
      │
      │   Anticodon loop
          │
          XXX ← ANTICODON (3 nucleotides complementary to mRNA codon)

• Smallest RNA (~73-95 nucleotides)
• Cloverleaf secondary structure
• L-shaped tertiary structure
• Acceptor stem (3' end: -CCA): Amino acid attachment site
  • Amino acid attached to 3'-OH of adenosine by aminoacyl-tRNA synthetase
• Anticodon loop: Recognizes mRNA codon by complementary base pairing
• DHU loop (Dihydrouracil loop): Aminoacyl-tRNA synthetase recognition
• TψC loop: Ribosome binding
• Contains many modified bases: Inosine, pseudouridine (ψ), dihydrouridine

• Adaptor molecule between mRNA codon and amino acid
• Carries activated amino acid to ribosome
• Decodes genetic information via anticodon-codon pairing

---

3. rRNA (Ribosomal RNA)

  EUKARYOTIC RIBOSOME (80S)
         │
    ┌────┴────┐
    ▼         ▼
  60S        40S
  subunit   subunit
    │            │
  28S rRNA    18S rRNA
  5.8S rRNA
  5S rRNA
  ~49 proteins  ~33 proteins
  
  
  PROKARYOTIC RIBOSOME (70S)
         │
    ┌────┴────┐
    ▼         ▼
  50S        30S
    │            │
  23S rRNA    16S rRNA
  5S rRNA

• Most abundant RNA (~80% of total RNA)
• Provides structural scaffold and catalytic activity of ribosomes
• 23S/28S rRNA has peptidyl transferase activity (a ribozyme)

---

4. hnRNA (Heterogeneous Nuclear RNA) / Pre-mRNA

  DNA ──TRANSCRIPTION──► Pre-mRNA (hnRNA)
                              │
                   ┌──────────┤
                   ▼          ▼
              5' CAPPING    3' POLY-A
                   │
                   ▼
             SPLICING
             (Removal of INTRONS)
             (Retaining EXONS)
                   │
                   ▼
              MATURE mRNA ──► Cytoplasm ──► Translation

---

5. snRNA (Small Nuclear RNA) and snRNP

• Components of spliceosome (splicing machinery)
• U1, U2, U4, U5, U6 snRNAs
• snRNA + proteins = snRNPs (snurps)
• Function: Recognition of splice sites; catalyze splicing of pre-mRNA introns

---

6. Regulatory RNAs

---

COMPARISON TABLE: mRNA vs tRNA vs rRNA

---

CENTRAL DOGMA (Context for RNA Functions):

  DNA ──[Replication]──► DNA

  DNA ──[Transcription]──► mRNA ──[Translation]──► Protein
                            ↑
                           rRNA (ribosome)
                           tRNA (adaptor)

  RNA ──[Reverse transcription]──► DNA
  (Retroviruses: HIV)

---

QUESTION 14: Define Enzymes. Classify with Suitable Examples. Add a Note on Mechanisms of Enzyme Action.

---

DEFINITION OF ENZYMES

• Thermodynamically, they lower the activation energy (Ea) of reactions
• They are highly specific (substrate specificity)
• They are regulated
• They are not altered at end of reaction

---

CLASSIFICATION OF ENZYMES (IUB/NC-IUBMB System - 7 Classes)

  ENZYMES
    │
    ├─ 1. OXIDOREDUCTASES
    │      (Oxidation-reduction reactions)
    │
    ├─ 2. TRANSFERASES
    │      (Transfer of functional groups)
    │
    ├─ 3. HYDROLASES
    │      (Hydrolysis reactions)
    │
    ├─ 4. LYASES
    │      (Addition/removal without water/oxidation)
    │
    ├─ 5. ISOMERASES
    │      (Isomerization reactions)
    │
    ├─ 6. LIGASES
    │      (Joining two molecules using ATP)
    │
    └─ 7. TRANSLOCASES (added 2018)
           (Movement across membranes)

Detailed Classification:

---

MECHANISMS OF ENZYME ACTION

Fundamental Concept - Activation Energy:

                         Transition State
                              ↑
  ENERGY                   ──/──
   LEVEL                  / ↑Ea (without enzyme)
                         /  |
  Substrates ───────────/   ↓Ea (with enzyme = LOWER)
                            |
  Products ─────────────────┘  ← Lower energy level
  
  Enzyme LOWERS Ea → Reaction faster
  Enzyme does NOT change ΔG (thermodynamics)

The Active Site:

  ENZYME
  ┌─────────────────────────────┐
  │                             │
  │    ┌─────────┐              │
  │    │ ACTIVE  │ ← Substrate  │
  │    │  SITE   │   binds here │
  │    └─────────┘              │
  │  (3D pocket / cleft)        │
  └─────────────────────────────┘
  
  Active site = Binding site + Catalytic site
  Made of few key amino acid residues
  (~3-10% of total enzyme volume)

Model 1: Lock and Key Model (Fischer, 1894)

  ENZYME            SUBSTRATE
  ┌──────┐          ┌──────┐
  │  KEY │    +     │ LOCK │ → Enzyme-Substrate Complex → Products
  │ SITE │          └──────┘
  └──────┘
  Rigid active site; exact geometric fit
  Limitation: Does not explain allosteric regulation

Model 2: Induced Fit Model (Koshland, 1958)

  Enzyme (open)    +    Substrate
  ┌──    ──┐            ┌──────┐
  │        │    ────►   │ │  │ │  ← Active site CHANGES SHAPE
  └──    ──┘            └──────┘     to fit substrate
  
  FLEXIBLE active site conforms to substrate
  Better explains: enzyme specificity, allosteric effects
  Currently accepted model

---

MECHANISMS OF CATALYSIS (How Enzymes Speed Up Reactions):

  CATALYTIC MECHANISMS
          │
    ┌─────┼──────┬──────┬──────┐
    ▼     ▼      ▼      ▼      ▼
  ACID-  COVAL-  METAL  PROXIMITY ELECTRO-
  BASE   ENT    ION    EFFECT  STATIC
  CATA-  CATA-  CATA-           STABI-
  LYSIS  LYSIS  LYSIS           LIZATION

---

Chymotrypsin Mechanism (Classic Example):

  Serine protease - uses CATALYTIC TRIAD (Asp102 - His57 - Ser195)

  Step 1: Substrate binds → His57 acts as base, accepts H+ from Ser195
  Step 2: Ser195-OH attacks peptide bond → Acyl-enzyme intermediate
  Step 3: Water molecule attacks → His57 activates water
  Step 4: Tetrahedral intermediate collapses → Product released
  Step 5: Enzyme regenerated

---

ENZYME KINETICS - MICHAELIS-MENTEN

  E + S ⇌ ES ──► E + P
  
  Km = [S] at which V = Vmax/2
       (Michaelis constant = measure of substrate affinity)
  
  Low Km = High affinity for substrate
  High Km = Low affinity for substrate
  
  Vmax = Maximum velocity (when all enzyme is saturated)

  1/V
   │
   │     /
   │    /
   │   /
   │  /
   │ /
   │/
───┼──────────────── → 1/[S]
   │
  -1/Km       y-intercept = 1/Vmax
              x-intercept = -1/Km

---

QUESTION 15: Define Enzymes. Explain in Detail Factors Affecting Enzyme Activity.

---

DEFINITION (same as Q14 - see above)

---

FACTORS AFFECTING ENZYME ACTIVITY

  FACTORS AFFECTING ENZYME ACTIVITY
               │
    ┌──────────┼──────────┬──────────┬──────────┐
    ▼          ▼          ▼          ▼          ▼
  SUBSTRATE  TEMPERATURE  pH      ENZYME    INHIBITORS
  CONCEN-                        CONCEN-   ACTIVATORS
  TRATION                        TRATION   COFACTORS

---

1. SUBSTRATE CONCENTRATION

  Velocity (V)
  ▲
  │         Vmax ─────────────────────────────── 
  │               ─────────────
  │          ────
  Vmax/2 ─ ─│─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ ─
  │         │
  └─────────┼──────────────────────────────────► [S]
            Km
  
  At low [S]: V increases proportionally (first order)
  At high [S]: V approaches Vmax (zero order)
  At [S] = Km: V = Vmax/2

• As [S] increases, V increases (hyperbolic curve)
• At saturation → Vmax (all enzyme molecules occupied)
• Km: Michaelis constant (measure of affinity)

---

2. TEMPERATURE

  Velocity (V)
  ▲
  │         ╱╲
  │        ╱  ╲ ← Enzyme denaturation
  │       ╱    ╲
  │      ╱      ╲
  │     ╱        
  └────────────────────────► Temperature (°C)
           ↑
       Optimum
      (37°C for
       most human
       enzymes)
  
  Below optimum: ↑ temp → ↑ reaction rate (10°C rule: Q10 = 2)
  Above optimum: Denaturation of enzyme → ↓ activity

• Q10 (temperature coefficient): Rate doubles for every 10°C rise
• Human enzymes: Optimum ~37°C
• Cold: Enzyme slows (preservation of food, cold storage of blood)
• High fever: Denatures some enzymes

---

3. pH

  Velocity (V)
  ▲
  │         /\
  │        /  \
  │       /    \
  │      /      \
  └───────────────────────► pH
          ↑
       Optimum
  
  Examples:
  Pepsin:       Optimum pH 1-2 (acid - stomach)
  Trypsin:      Optimum pH 7-8 (alkaline - intestine)
  Salivary amylase: Optimum pH 6.8-7.0
  Alkaline phosphatase: Optimum pH 9-10

• pH affects ionization of amino acid residues in the active site
• pH affects substrate ionization
• Extreme pH → Denaturation
• Each enzyme has characteristic optimum pH

---

4. ENZYME CONCENTRATION

  Velocity (V)
  ▲
  │         /
  │        /
  │       /   (Linear relationship)
  │      /
  │     /
  └──────────────────────► [Enzyme]
  
  At constant [S] >> Km: V is directly proportional to [E]

• More enzyme molecules → more substrate processed per unit time
• Basis of enzyme assays in clinical labs (measure enzyme concentration indirectly)

---

5. COFACTORS AND COENZYMES

  APOENZYME (inactive protein)
       +
  COFACTOR
       =
  HOLOENZYME (active)
       │
    ┌──┴──────────┐
    ▼             ▼
 INORGANIC     ORGANIC
 IONS          (Coenzymes)
(Mg²⁺, Zn²⁺,  (NAD+, FAD,
Fe²⁺, Cu²⁺)    CoA, PLP)

---

6. ALLOSTERIC REGULATION (Metabolic Control)

  ALLOSTERIC ENZYME
  ┌─────────────────────────────────────┐
  │  ACTIVE   │         │  ALLOSTERIC  │
  │   SITE    │         │    SITE      │
  └─────────────────────────────────────┘
                              ↑
                    Modulator binds here
                    (at site different from active site)
                              │
                    ┌─────────┴──────────┐
                    ▼                    ▼
              POSITIVE              NEGATIVE
              ALLOSTERIC           ALLOSTERIC
              EFFECTOR             EFFECTOR
              (Activator)          (Inhibitor)
              e.g., AMP            e.g., ATP
              activates PFK-1      inhibits PFK-1

  V
  ▲  Allosteric enzyme
  │           ___
  │         /
  │       /  ← Cooperative binding
  │     /     (sigmoidal)
  └──────────────────► [S]
  
  Hill coefficient (n) > 1 = positive cooperativity

---

7. INHIBITORS (briefly - see Q16 for full detail)

• Competitive: Increase [S] overcomes inhibition
• Non-competitive: Increase [S] does NOT overcome inhibition
• Irreversible: Permanent inactivation

---

8. HORMONAL AND FEEDBACK REGULATION

  FEEDFORWARD ACTIVATION:
  Glucose-6-phosphate ──► activates Glycogen synthase
  
  FEEDBACK INHIBITION:
  End product ──► inhibits first enzyme of pathway
  
  Example:
  Threonine ──► Threonine deaminase ──► Isoleucine
                     ↑
              Inhibited by Isoleucine (end product)
              (Negative feedback)

---

QUESTION 16: What is Enzyme Inhibitor? Give Detail Account of Types of Enzyme Inhibitions with Their Clinical Significance.

---

DEFINITION OF ENZYME INHIBITOR

---

CLASSIFICATION OF ENZYME INHIBITION

  ENZYME INHIBITION
         │
    ┌────┴──────────┐
    ▼               ▼
REVERSIBLE      IRREVERSIBLE
    │
    ├──► COMPETITIVE
    ├──► NON-COMPETITIVE
    ├──► UNCOMPETITIVE
    └──► MIXED

---

1. COMPETITIVE INHIBITION

  MECHANISM:
  
  Inhibitor (I) RESEMBLES substrate (S)
  Competes for the SAME active site
  
  Normal:    E + S ──► ES ──► E + P
  Inhibited: E + I ──► EI (dead-end complex, no product)
  
  ┌─────────┐        ┌─────────┐
  │  ENZYME │        │  ENZYME │
  │ ┌─────┐ │   OR   │ ┌─────┐ │
  │ │  S  │ │        │ │  I  │ │
  │ └─────┘ │        │ └─────┘ │
  └─────────┘        └─────────┘
  Active form         Inhibited

• Km increases (↓ apparent affinity)
• Vmax UNCHANGED (can be overcome by ↑ substrate)
• Inhibition is REVERSIBLE

  1/V
   │            / Inhibited (steeper slope)
   │           /
   │          / Normal
   │         /
   │────────────────────────────── → 1/[S]
   Same y-intercept (1/Vmax)
   Different x-intercept (Km changes)

---

2. NON-COMPETITIVE INHIBITION

  MECHANISM:
  
  Inhibitor binds ALLOSTERIC SITE (not active site)
  Can bind FREE enzyme OR ES complex
  
  E + I ──► EI (inactive)
  ES + I ──► ESI (inactive)
  
  Substrate can STILL bind, but catalysis blocked
  
  ┌─────────────────────┐
  │  ENZYME             │
  │ ┌─────┐  ┌───────┐  │
  │ │  S  │  │   I   │  │ ← Binds allosteric site
  │ └─────┘  └───────┘  │   while S still in active site
  └─────────────────────┘

• Km UNCHANGED (substrate still binds normally)
• Vmax DECREASES (cannot be overcome by ↑ substrate)

  1/V
   │         / Inhibited (↑ y-intercept)
   │        /
   │       / Normal
   │      /
   └──────────────────────────────► 1/[S]
   Same x-intercept (Km same)
   Different y-intercept (Vmax changes)

• Heavy metals (Pb²⁺, Hg²⁺, As³⁺) - bind -SH groups
• Lead poisoning: Inhibits δ-aminolevulinic acid dehydratase (ALAD) and ferrochelatase → accumulation of δ-ALA, zinc protoporphyrin → anemia, neurological symptoms

---

3. UNCOMPETITIVE INHIBITION

  MECHANISM:
  
  Inhibitor binds ONLY to ES complex (NOT free enzyme)
  
  E + S ──► ES + I ──► ESI (inactive, NO product)
  
  BOTH Km and Vmax decrease (in same proportion)
  Km (apparent) DECREASES

• Km decreases (apparent)
• Vmax decreases
• Lines PARALLEL on Lineweaver-Burk plot

---

4. MIXED INHIBITION

• Inhibitor can bind both free enzyme and ES complex, but with different affinities
• Both Km and Vmax are altered
• When binding affinities are equal → Pure non-competitive inhibition

---

5. IRREVERSIBLE INHIBITION

  MECHANISM:
  
  Inhibitor forms a COVALENT BOND with enzyme
  Enzyme is PERMANENTLY inactivated
  Cannot be reversed by dialysis or dilution
  
  E ──── I  (covalent, stable)
  (enzyme destroyed)
  
  Suicide inhibitors: Converted by enzyme to reactive form
  that then irreversibly inhibits

---

COMPARISON TABLE OF INHIBITION TYPES

---

ALLOSTERIC INHIBITION (Metabolic Regulation)

  FEEDBACK INHIBITION PATHWAY:

  A ──E1──► B ──E2──► C ──E3──► D (end product)
  ↑
  │ D inhibits E1 (first/committed step)
  └─────────────────────────────────────
  
  Example: Isoleucine inhibits threonine deaminase
           CTP inhibits aspartate transcarbamoylase (ATCase)

---

QUESTION 17: Write in Detail Diagnostic and Therapeutic Importance of Enzymes.

---

ENZYMES AS DIAGNOSTIC TOOLS

  ORGAN DAMAGE / DISEASE
         │
         ▼
  Cell membrane disruption
         │
         ▼
  Intracellular enzymes leak into blood
         │
         ▼
  ↑ Serum enzyme activity
         │
         ▼
  Diagnostic information:
  - Which organ is damaged?
  - How severe is the damage?
  - Is the patient recovering?

---

DIAGNOSTIC ENZYMES - BY ORGAN/DISEASE

1. CARDIAC ENZYMES (Myocardial Infarction)

  TIME COURSE AFTER MI:
  
  Enzyme level
  ▲   CK-MB / Troponin
  │         ╱─────╲      Troponin stays elevated longest
  │        /         \___
  │   CK-MB           Troponin I/T
  │  ╱       ╲
  │ ╱          ╲___________
  └──────────────────────────────► Hours after MI
     0  6  12  24  48  72  120

• CK-MM: Skeletal muscle (95% of total CK)
• CK-MB: Heart muscle (> 6% of total CK in MI)
• CK-BB: Brain

• LDH1 > LDH2: MI ("flipped LDH")
• LDH5 > LDH4: Liver disease

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2. LIVER ENZYMES (Hepatic Disease)

  AST/ALT < 1 → Viral hepatitis (ALT > AST)
  AST/ALT > 2 → Alcoholic hepatitis
  AST/ALT > 3 → Strongly suggests alcoholic liver disease

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3. PANCREATIC ENZYMES (Pancreatitis)

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4. BONE AND PROSTATE ENZYMES

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5. HEMOLYTIC/GENETIC DISORDERS

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6. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

  ENZYME AS DIAGNOSTIC TOOL (ELISA):
  
  ANTIGEN (sample)
       │
       ▼
  Capture antibody (solid phase)
       │
       ▼
  Detection antibody (enzyme-linked)
       │  [HRP or Alkaline Phosphatase]
       ▼
  ADD SUBSTRATE
       │
       ▼
  COLORED PRODUCT (absorbance measured)
       │
       ▼
  Quantify analyte (hormone, HIV antigen, etc.)

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THERAPEUTIC USES OF ENZYMES

1. THROMBOLYTIC (FIBRINOLYTIC) THERAPY

  CORONARY/PULMONARY THROMBOSIS
               │
               ▼
  STREPTOKINASE / UROKINASE / tPA (Tissue Plasminogen Activator)
               │
               ▼
  Activates plasminogen → PLASMIN
               │
               ▼
  Plasmin cleaves FIBRIN
               │
               ▼
  CLOT DISSOLVED (Thrombolysis)

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2. DIGESTIVE ENZYME SUPPLEMENTS

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3. ENZYME REPLACEMENT THERAPY (ERT) - Lysosomal Storage Diseases

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4. ANTI-INFLAMMATORY / WOUND HEALING

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5. ANTI-CANCER ENZYME THERAPY

  L-ASPARAGINASE
       │
       ▼
  Converts L-Asparagine ──► Aspartate + NH₃
       │
  Normal cells: Can synthesize asparagine (asparagine synthetase)
  Leukemic cells: CANNOT synthesize asparagine (asparagine synthetase-deficient)
       │
       ▼
  Asparagine depletion → Leukemic cell DEATH
       │
       ▼
  TREATMENT OF ALL (Acute Lymphoblastic Leukemia)

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6. CARDIOVASCULAR AND OTHER USES

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SUMMARY FLOWCHART - ENZYME USE IN MEDICINE

  ENZYMES IN MEDICINE
         │
    ┌────┴────────────────────────────────┐
    ▼                                     ▼
DIAGNOSTIC                          THERAPEUTIC
    │                                     │
    ├── Marker of organ damage            ├── Thrombolytics (tPA, streptokinase)
    │   (AMI: Troponin, CK-MB)           ├── Digestive enzymes (pancrelipase)
    │   (Liver: ALT, AST, ALP)           ├── ERT (Gaucher's, Pompe's)
    │   (Pancreas: Amylase, Lipase)      ├── Anti-cancer (L-asparaginase)
    │                                     ├── Wound debridement (collagenase)
    ├── ELISA (HIV, hormones)             └── Gene therapy/recombinant enzymes
    │
    └── Enzyme assays for
        genetic diseases (G6PD,
        Hexosaminidase A)

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Exam Tips Summary:

• Q11: Always draw the DNA double helix with dimensions (3.4 Å, 34 Å, 10 bp/turn). AZT = chain terminator (no 3'-OH) is a must-know.
• Q12: B/A/Z-DNA table is high-yield. Biologically important nucleotides: ATP (energy), cAMP (second messenger), NAD+ (coenzyme), UDP-glucose (glycogen synthesis).
• Q13: Draw the tRNA cloverleaf. mRNA cap = 7-methylguanosine. Polycistronic = prokaryote only.
• Q14: Lock and key (Fischer) vs Induced fit (Koshland) - Induced fit is currently accepted. Catalytic triad = Asp-His-Ser in serine proteases.
• Q15: Draw all 4 graphs (substrate, temperature, pH, enzyme concentration). Allosteric enzymes show sigmoidal kinetics.
• Q16: Aspirin = irreversible COX inhibitor (acetylates Ser). Statins = competitive inhibitors of HMG-CoA reductase. Organophosphates = irreversible AChE inhibitors.
• Q17: Troponin T/I = gold standard for MI (most sensitive + specific). tPA = treatment of ischemic stroke within 4.5 hours. L-Asparaginase = treatment of ALL.

MBBS Biochemistry Q11–17 — Quick Summary

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Q11 — Nucleotide, Nucleoside & DNA Double Helix

Key facts to remember:
• Right-handed helix
• Antiparallel strands (5'→3' / 3'→5')
• A=T (2 H-bonds) | G≡C (3 H-bonds)
• 10 bp/turn | Rise = 3.4 Å | Pitch = 34 Å
• Chargaff's rule: A=T, G=C

• Anticancer: 5-FU (inhibits thymidylate synthase), 6-MP, Cytarabine, Methotrexate
• Antiviral: AZT (HIV - chain terminator, no 3'-OH), Acyclovir (Herpes), Sofosbuvir (HCV)

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Q12 — DNA Conformations + Biologically Important Nucleotides

ATP       → Energy currency
cAMP/cGMP → Second messengers
NAD+/FAD  → Coenzymes (H carriers)
UDP-Glucose → Glycogen synthesis
GTP       → Protein synthesis, G-proteins
SAM       → Methyl donor

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Q13 — Types, Structure & Functions of RNA

RNA TYPES
├── mRNA  → Carries genetic code | 5'cap + PolyA tail
├── tRNA  → Adaptor (cloverleaf) | Anticodon binds codon
├── rRNA  → Ribosome (80S eukaryote; 70S prokaryote)
├── hnRNA → Pre-mRNA; undergoes splicing
├── snRNA → Spliceosome (U1,U2,U4,U5,U6)
└── miRNA/siRNA → Gene silencing (RNAi)

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Q14 — Enzymes: Classification & Mechanism

1. Oxidoreductases  → Oxidation/reduction (LDH, Cytochrome oxidase)
2. Transferases     → Group transfer (Kinases, Aminotransferases)
3. Hydrolases       → Hydrolysis (Trypsin, Amylase, Lipase)
4. Lyases           → C-C/C-O breaking (Aldolase, Fumarase)
5. Isomerases       → Rearrangement (Phosphoglucose isomerase)
6. Ligases          → Join molecules + ATP (DNA ligase, Pyruvate carboxylase)

Substrate → [Lowers Activation Energy] → Product

Models:
• Lock & Key (Fischer 1894) — rigid active site
• Induced Fit (Koshland 1958) — flexible active site [ACCEPTED]

Catalytic strategies:
Acid-Base | Covalent | Metal ion | Proximity/Orientation

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Q15 — Factors Affecting Enzyme Activity

FACTOR          EFFECT
──────────────────────────────────────────────────
[Substrate] ↑   V increases → plateau at Vmax
Temperature ↑   V increases until optimum (37°C), then ↓ (denaturation)
pH change       Alters ionization of active site residues; each enzyme has optimum
[Enzyme] ↑      V increases linearly (at saturating [S])
Cofactors       Apoenzyme + Cofactor = Holoenzyme (active)
Allosteric reg. AMP activates PFK-1; ATP inhibits PFK-1 (sigmoidal curve)
Inhibitors      Competitive / Non-competitive / Irreversible

• Low Km = High affinity
• High Km = Low affinity

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Q16 — Enzyme Inhibition

TYPE           Km        Vmax    Reversible?  Key Example
──────────────────────────────────────────────────────────
Competitive    ↑ (↑)     Same    YES          Statins (HMG-CoA reductase)
Non-competitive Same      ↓       YES          Heavy metals (Pb²⁺, Hg²⁺)
Uncompetitive  ↓         ↓       YES          Li⁺ (inositol monophosphatase)
Irreversible   —         ↓↓      NO           Aspirin (COX), Organophosphates (AChE)

• Aspirin → Irreversible COX inhibitor → antiplatelet
• Statins → Competitive inhibitor of HMG-CoA reductase → ↓ cholesterol
• Organophosphates → Irreversible AChE inhibition → ACh accumulation → SLUDGE
• Methotrexate → Competitive inhibitor of DHFR → ↓ THF → cancer/RA
• Omeprazole (PPI) → Irreversible H⁺/K⁺ ATPase inhibitor → peptic ulcer
• Penicillin → Irreversible transpeptidase inhibitor → antibiotic

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Q17 — Diagnostic & Therapeutic Importance of Enzymes

Diagnostic:

ORGAN        ENZYME MARKER           SIGNIFICANCE
────────────────────────────────────────────────────────────
Heart (MI)   Troponin I/T (GOLD STD) Most sensitive & specific
             CK-MB                   MI (rises 4-8h, peaks 24h)
Liver        ALT > AST               Viral hepatitis
             AST/ALT > 2             Alcoholic hepatitis
             ALP + GGT               Cholestasis
Pancreas     Lipase (best)           Acute pancreatitis (specific)
             Amylase                 Acute pancreatitis (fast, less specific)
Prostate     PSA                     Prostate cancer
Hemolysis    G6PD assay              G6PD deficiency

Therapeutic:

USE                  ENZYME DRUG              CONDITION
──────────────────────────────────────────────────────────────
Thrombolysis         tPA (Alteplase)          Stroke (within 4.5h), MI
                     Streptokinase            MI, PE, DVT
Digestion            Pancrelipase             Cystic fibrosis, pancreatitis
ERT                  Imiglucerase             Gaucher's disease
                     Alglucosidase alfa       Pompe's disease
Anti-cancer          L-Asparaginase           ALL (leukemia)
Mucus clearance      Dornase alfa (DNase)     Cystic fibrosis
Wound debridement    Collagenase              Burns, wounds
Tumor lysis          Rasburicase              Hyperuricemia

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MASTER MNEMONICS