Q.45 Given below are two statements : One is labelled as Assertion A and the other is labelled as Reason R
Assertion A : While the pKa of a free lysine is 10.5, the pKa of lysine decreases to 6.6 when present in the
active site of Acetoacetate decarboxylase.
Reason R : The decreased pKa of lysine in the active site is due to the electrostatic effect of nearby positive
charges.
In the light of the above statements. choose the most appropriate answer from the options given below :
1. Both A and R are correct and R is the conect explanation of A
2. Both A and R are correct but R is NOT the correct explanation of A
3. A is conect but R is not correct
4. A is not correct but R is correct
Correct Answer: 3. A is correct but R is not correct
In acetoacetate decarboxylase, a key lysine residue in the active site has its pKa shifted from 10.5 (free) to ~6.0-6.6 to act as a nucleophile, but this lowering occurs due to a nearby negative microenvironment (not positive charges), enabling protonated Lys at physiological pH.
Assertion (A) Analysis
Assertion A is correct. Free lysine’s ε-amino group has pKa ≈10.5 (protonated below, neutral above). In acetoacetate decarboxylase (AAD), active site Lys-115 pKa drops to ~6.6, confirmed by kinetic/pH studies. This allows deprotonation near neutral pH, forming a covalent Schiff base with acetoacetate substrate for decarboxylation.
Reason (R) Analysis
Reason R is incorrect. A pKa decrease for lysine (weaker base, easier deprotonation) requires stabilization of the deprotonated (neutral) form. Nearby positive charges would destabilize this neutral amine via repulsion, raising pKa. In AAD, a hydrophobic pocket and nearby Asp/Glu carboxylates (negative) stabilize the neutral ε-NH₂ via H-bonding or desolvation, lowering pKa.
Option Breakdown
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Option 1: Both correct, R explains A—False. R’s mechanism wrong.
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Option 2: Both correct, R not explanation—False. R incorrect.
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Option 3: A correct, R incorrect—True. pKa values match; explanation reversed.
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Option 4: A incorrect, R correct—False. A experimentally verified.
Lysine pKa active site acetoacetate decarboxylase shift enables catalysis, correctly stated in Assertion A but wrongly explained in Reason R—vital for enzyme mechanism questions in competitive exams.
Normal vs Enzyme Lysine pKa
Free Lys: pKa 10.5 → protonated (LysH⁺) at pH 7.
AAD Lys-115: pKa ~6.6 → ~50% deprotonated (Lys-NH₂) at pH 7, nucleophilic for Schiff base.
Mechanism: Lys-NH₂ attacks acetoacetate carbonyl → imine → decarboxylation → protonation cycle.
Why pKa Lowers: Correct Explanation
pKa shift ΔpKa = -4 requires ΔG ~5 kcal/mol stabilizing neutral form:
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Hydrophobic microenvironment: Desolvates charged LysH⁺ more than neutral NH₂.
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Negative charges nearby (Asp/Glu): H-bond neutral ε-NH₂; repel protonated form.
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No water: Rigid helix holds conformation.
R Error: Positive charges (Arg/Lys) raise pKa by repelling electrons from ε-NH₂.
| Factor | Effect on Lys pKa | AAD Example |
|---|---|---|
| Nearby + (Arg) | ↑ pKa (destabilizes NH₂) | Not present |
| Nearby – (Asp) | ↓ pKa (stabilizes NH₂) | Key residue |
| Hydrophobic | ↓ pKa (desolvation) | Active site pocket |
Exam Application
Tests biophysical chemistry: pKa shifts follow charge logic (opp. charges lower pKa for bases). Common trap—confusing stabilization of conjugate base vs. acid. Essential for GATE BT/LS enzyme kinetics.
