Chapter 5 : Enzyme

Multiple Choice Questions – Core Ideas, Traps & Teacher Tips

Q1 Starch + Amylase (25 °C, 1 h)

• Experimental set-up
  • 2 % starch (amylose) mixed with 2 % amylase, incubated 60 min at $25\,^{\circ}\text{C}$.
  • Samples tested with Biuret, Benedict’s (with heat), Iodine in KI.
• Key chemical facts
  • Amylase is a protein ⇒ gives a positive Biuret (purple) if not too dilute.
  • Products of starch hydrolysis = maltose (major) + traces of glucose; both are reducing sugars ⇒ Benedict’s turns brick-red after complete hydrolysis.
  • If all starch digested, iodine test is negative (yellow-orange) because no helical amylose remains to form the blue-black complex.
• Expected colour set therefore = Biuret: purple, Benedict’s: brick red, Iodine: orange ⇒ Option D.
• Pedagogical notes
  • Always ask (i) “What is the reagent testing for?” (ii) “Is the analyte present after reaction?”

Q2 pH reversibility & Protein Levels

• Given: single polypeptide chain, reversible loss of activity when pH lowered slightly.
• Reasoning path
  • Single chain ⇒ no quaternary structure; eliminate C.
  • Primary structure & covalent peptide bonds are unaffected by mild pH change (would be irreversible) ⇒ eliminate A & D.
  • Mild pH alters ionisation of R-groups ⇒ disrupts ionic / H-bonds → slight tertiary structure distortion, reversible when pH restored ⇒ Option B.

Q3 Active-site Residues in Phospholipase

• Stated: only His311 & His356 take part in catalysis.
• Logic
  • His356 is explicitly catalytic.
  • Tyr551 contacts substrate without chemistry (forms H-bond/van der Waals) ⇒ contact residue.
  • Cys570 forms disulfide stabilising tertiary fold ⇒ structural residue.
• Pattern matches Option C.

Q4 How Enzymes Act

• Eliminate distractors
  • A Cofactors may participate at active site or elsewhere; wording too narrow.
  • B Permanent covalent attachment would trap product; typical bonds are weak & reversible.
  • C Induced-fit: major conformational change for enzyme; substrate only slightly distorted → statement partially wrong.
  • D Most enzyme reactions are reversible; enzyme usually binds product with lower affinity, not zero.
• The best statement among the four is therefore None (if forced, C is closest but still wrong – highlights need to read wording carefully).

Q5 Why Amylase Cannot Hydrolyse Cellulose

• Cellulose = $\beta\text{(1→4)}$ glycosidic bonds vs starch $\alpha\text{(1→4)}$ & $\alpha\text{(1→6)}$.
• Difference in stereochemistry changes 3-D shape ⇒ enzyme specificity lost.
• Correct MCQ answer D.

Q6 Substrate Concentration vs Time Graph

• Requirement: [S] must decrease monotonically & asymptotically approach zero.
• Theoretical curve: steep fall initially (fast rate), then tail as substrate exhausted ⇒ matches Graph D.

Q7 Comparing 5 Temperature & pH Curves

• Most plausible explanation options
  • A Hydrogen bonds disrupted at 60 °C ⇒ explains loss of activity of enzyme 2 ⇒ Correct.
  • Others contain conceptual errors (wrong pH charge state, unsupported ‘hydrogen bonds only’, kinetic energy highest at 90 °C not 75 °C).

Q8 Two Temperature Experiments (37 °C vs ramp to 80 °C)

• Key expectations
  • Experiment Y (to 80 °C) has steeper initial slope (higher KE) but lower final product (denaturation).
• Chosen graph A.

Q9 Metabolic Pathway to Lysine

• Strategy for MCQ
  • Identify branch or feedback steps, ask: “Which change alleviates bottleneck longest?”
  • Increasing enzyme 3 (downstream) shunts more flux to lysine without depleting precursor for other pathways ⇒ Option C.

Q10 Herbicide Resembling Glutamate

• Structural mimic → competes for active site of glutamine synthetase ⇒ Competitive inhibitor (A).

Q11 Effect of Increasing [S] on Inhibition

• Competitive inhibition can be overcome (decreases), non-competitive unchanged.
• Table row B (competitive ↓, non-competitive same).

Structured / Data-handling Questions – Examiner Mark Scheme Highlights

Ant Speed vs Temperature (2021 P2 Q2)

• Description template
  • Quote two regions (9–31.5 °C gradual rise to 4 cm s$^{-1}$; 31.5–38.5 °C sharp rise to 6.8 cm s$^{-1}$).
• Explanation
  • Higher T ⇒ ↑KE ⇒ ↑freq successful E–S collisions in muscle & respiration enzymes ⇒ ↑ATP ⇒ ↑speed.
• Prediction above 40 °C
  • Increases to optimum, then falls to 0 as enzymes denature (H-bonds break, active site lost).

Enzyme Thermostability Graphs (2018 P2 Q2)

• Thermitase: steep rise to 76 °C (650 a.u.), then sharp decline.
• Denaturation above 80 °C disrupts H-bonds & hydrophobic core.
• Modified subtilisin (8 AA replaced) vs subtilisin
  • Higher Vmax (320 vs 80 a.u.) & higher $T_{opt}$ (76 vs 59 °C).
  • Likely more disulfide bonds / stronger ionic interactions → more thermostable OR better active-site fit.

Effect of [S] with/without Inhibitors (2016 P2 Q1; 2011 P2 Q1)

• “Enzyme only” curve hyperbolic (Michaelis-Menten), plateau when active sites saturated.
• Non-competitive inhibitor lowers $V<em>{max}$, unchanged $K</em>m$.
• Competitive inhibitor raises apparent $K<em>m$ but reaches same $V</em>{max}$ at high [S]. Sketch passes through enzyme-only curve at high [S].

pH Optimum Example – Chymosin (2002 P2 Q6)

• Optimum pH 3.
• Alkaline pH 8 deprotonates acidic R-groups, breaks ionic/H-bonds, denatures active site ⇒ activity lost.

Core Theory & Explanatory Notes

Hierarchical Protein Structure

• Primary: linear amino-acid sequence joined by peptide bonds $\text{(–CO–NH–)}$.
• Secondary: $\alpha$-helix & $\beta$-sheet stabilised by H-bonds between $C=O$ and $N–H$ of backbone.
• Tertiary: overall 3-D fold held by
  • Hydrogen bonds
  • Ionic (salt-bridge) bonds
  • Disulfide (covalent $S–S$) bonds
  • Hydrophobic interactions (non-polar side chains cluster).
• Quaternary: assembly of ≥2 polypeptides, same bond types but intermolecular.

Active Site & Catalysis Mechanisms

• Specific 3-D pocket with catalytic & contact residues.
• Lock-and-Key vs Induced-Fit
  • Induced fit: substrate binding prompts conformational tightening, enhances complementarity.
• Ways enzymes lower $E_A$
  1. Proximity/orientation: hold substrates in correct alignment.
  2. Transition-state stabilisation: complementary to high-energy state.
  3. Bond strain: distort substrate bonds.
  4. Acid–base & covalent catalysis (temporary covalent bond with catalytic residue).

Kinetics & Michaelis–Menten Parameters

• Initial rate $v$ vs [S] produces hyperbola.
• $V_{max}$: plateau when enzyme saturated.
• $K<em>m$: [S] giving $v = V</em>{max}/2$ (measure of affinity, lower $K_m$ ⇒ higher affinity).

Factors Affecting Enzyme Activity

1. Temperature
   • Below $T_{opt}$: rate ∝ KE (≈ Q10 rule: rate doubles every 10 °C).
   • Above $T_{opt}$: disruption of weak bonds → denaturation.
2. pH
   • Alters protonation state of acidic/basic residues; extremes disrupt ionic & H-bonds.
3. Substrate Concentration
   • Low [S]: first-order w.r.t [S].
   • High [S]: zero-order (rate independent of [S]).
4. Enzyme Concentration: linear with rate when [S] saturating.
5. Cofactors / Coenzymes
   • Metal ions (Zn$^{2+}$, Fe$^{2+}$) or organic molecules (NAD$^{+}$, FAD) essential for activity.

Inhibition Types

• Competitive
  • Binds active site; resembles substrate.
  • Raises apparent $K<em>m$, same $V</em>{max}$.
  • Effect decreases as [S]↑.
• Non-competitive (allosteric)
  • Binds elsewhere; alters active site.
  • Lowers $V<em>{max}$, $K</em>m$ unchanged.
  • Not relieved by high [S].
• Uncompetitive, Mixed & Irreversible (e.g. penicillin) – advanced examples.

Metabolic Pathway Control

• Feedback inhibition: end-product binds allosterically to first committed enzyme.
• Branch-point enzymes regulate distribution of precursors.
• Increasing activity downstream (e.g. enzyme 3 in lysine pathway) enhances flux to desired product without accumulating toxic intermediates.

Thermostable & Extremophile Enzymes (e.g. Taq Polymerase)

• Additional disulfide/ionic bonds, tighter hydrophobic core, shorter surface loops.
• Applications: PCR cycles 95 °C denaturation yet enzyme remains functional.

Importance of Protein Size (>100 AA) for Function

• Provides sufficient length to fold into stable catalytic/ligand-binding shapes.
• Transmembrane receptors need seven $\alpha$-helices + extracellular & cytoplasmic loops (≈300 AA).
• Enzymes often require domains for substrate, cofactor, regulation.

Worked Explanations & Model Phrases (Exam Technique)

• When DESCRIBE graph: quote data with units, reference regions, use qualifiers (“gradually”, “steeply”).
• When EXPLAIN rate change: link physical parameter → molecular effect → collision frequency → E-S complex → rate.
• Use term “enzyme-substrate complex (E–S)” once defined.
• Avoid saying “produce energy”; correct = “produce ATP”.

Numerical / Statistical Reminders

• Reaction rate units often $\text{mol dm}^{-3}\text{ s}^{-1}$ or arbitrary units (a.u.).
• Speed example: $6.8\,\text{cm s}^{-1}$ at $38.5\,^{\circ}\text{C}$.
• Optimum temperature example: Thermitase $76\,^{\circ}\text{C}$.
• Optimum pH example: Chymosin pH 3.

Ethical & Practical Notes

• Use of calf chymosin raises animal welfare & GMO alternatives (recombinant rennin) in cheese industry.
• Herbicide targeting glutamine synthetase may accumulate ammonia → ecological impact on non-target plants.

Quick-fire Revision Points

• Amylase hydrolyses $\alpha(1→4)$ linkages only.
• Subtilisin modification improves detergent enzymes for high-temp washing.
• Penicillin mimics D-ala-D-ala cell-wall peptide; irreversible suicide inhibitor of transpeptidase.
• Q10 ≈ 2 for many enzymes between 0–40 °C.
• $E_A$ lowered but $\Delta G$ of reaction unchanged.

Exam Tip Checklist

• ALWAYS state units when quoting numeric data.
• Differentiate ‘rate increases UNTIL optimum’ vs ‘activity maximum AT optimum’.
• For inhibitors, mention binding site AND effect on $V<em>{max}$/$K</em>m$.
• Sketches: include initial slope, plateau, relative positions.
• In essays, cover at least two different protein examples for shape-function arguments (enzyme, receptor, structural, transport).