Enzymes as Catalysts – Comprehensive Study Notes

Page 1 – Course & Reading Context

• Lecture title: “Enzymes as Catalysts I.”
• Instructor: Dr. Amy M. Hicks, PhD, MPH – Biochemistry Course Director (VCOM–Carolinas).
• Assigned reading: Lieberman & Peet, Biochemistry, Ch. 8 – foundational chapter that describes enzyme catalysis, kinetics, active‐site architecture, and physiological significance.
• Pedagogic framing: Positioned early in the medical‐biochemistry sequence; links molecular understanding of enzymes to systemic physiology, pathology, and pharmacology.

Page 2 – Myoglobin vs. Hemoglobin Oxygen Affinity

1. Higher affinity?
   • $\text{Mb} > \text{Hb}$. Myoglobin binds O₂ more tightly (hyperbolic curve) than hemoglobin (sigmoidal/co-operative).
2. Problem if Hb had Mb-like high affinity:
   • Hb would not release sufficient O₂ to tissues – systemic hypoxia despite normal arterial $\text{pO}_2$.
3. Evidence of co-operativity:
   • Sigmoidal $\text{Hb‐O}<em>2$ dissociation curve displays an inflection – slope steepens after first O₂ binding (Hill coefficient $n</em>H>1$).
4. Hb affinity at varying $\text{pO}_2$:
   • Lungs (≈100 torr): near-maximal saturation → efficient loading.
   • Resting tissue (≈30 torr): ~60 % saturation → partial unloading.
   • Highly active tissue (≈3 torr): dramatic unloading → steep part of curve ensures O₂ delivery where most needed.

• Physiological connection: Cooperative binding allows a single protein (Hb) to serve both roles—loading and unloading—through an allosteric T⇌R equilibrium shift modulated by pO₂, pH, CO₂, and 2,3-BPG.

Page 3 – Factors Promoting O₂ Dissociation (Bohr Effect & More)

• Lower pH (Bohr effect): Protonation of specific residues stabilizes the T‐state → ↓ affinity.
• Carbamino formation (CO₂ bound to Hb N-termini):
  • Reaction: $\text{R–NH}<em>2 + \text{CO}</em>2 \rightarrow \text{R–NH–COO}^- + \text{H}^+$.
  • Adds negative charge ↔ salt bridges that favor deoxy conformer.
• 2,3-BPG binding: Occupies central cavity of deoxy Hb; electrostatic interactions lock T‐state.
• CO₂ concentration: Elevated tissue CO₂ → more H⁺ and carbamate formation → enhanced O₂ unloading.

Page 4 – pH, CO₂ Hydration & Physiological Relevance

• Reaction in blood: $\text{CO}<em>2 + \text{H}</em>2\text{O} \rightleftharpoons \text{HCO}_3^- + \text{H}^+$.
• Physiological context: In metabolically active tissue, ↑CO₂ drives reaction rightward → ↓pH → Bohr shift.
• Conversely in lungs, low CO₂ pulls reaction leftward, raising pH and favoring Hb R‐state.

Page 5 – Role of Carbonic Anhydrase (CA)

• Key enzyme: CA in RBCs accelerates above reaction by >10⁶-fold; essential because uncatalyzed hydration is too slow for respiratory demand.
• No true carbonic acid intermediate; CA uses a Zn²⁺-activated hydroxide to attack CO₂.
• Exchange: $\text{HCO}_3^-$ exits RBC via anion exchanger (Band 3) ↔ Cl⁻ shift; some H⁺ binds Hb.
• In lungs: Process reverses (low CO₂) → bicarbonate re-enters, reforms CO₂ for exhalation.

Page 6 – Carbamate Pathway for CO₂ Transport

• CO₂ + terminal α‐amino on each Hb chain → carbamate.
• Effect: Adds –ve charge, forms additional salt bridges → stabilizes T‐state (deoxy).
• Reversibility: Dissociates in pulmonary capillaries (high O₂, low CO₂) restoring R‐state and releasing CO₂.

Page 7 – Submarine CO₂ Accumulation Question (Concept Check)

If ambient CO₂ rises:
• Hb will have lower O₂ affinity (right-shifted curve) owing to Bohr effect & carbamino formation.
– Correct MCQ option: “Their hemoglobin will have a lower oxygen affinity…”
• Mb affinity largely unaffected (inside muscle), CA does not ‘cease,’ bicarbonate would actually increase.

Page 8 – Lecture Objectives (Comprehensive List)

A. Explain enzymes as essential biological catalysts enabling life-compatible reaction rates.
B. Correlate enzyme deficiency/overactivity with disease; recognize drug design strategies exploiting enzymes.
C. Describe active-site architecture, substrate selectivity, transition-state stabilization.
D. Analyze how [E], [S], T, pH govern rate (Michaelis–Menten, Arrhenius, ionization).
E. Identify six major enzyme classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases.
F. Interpret free-energy change $\Delta G^o$ for spontaneity.
G. Draw energy coordinate diagram; distinguish $\Delta G^o$ vs. $\Delta G^{\ddagger}$.
H. Provide pharmacological example (e.g., aspirin inhibiting COX-1; prodrugs).
I. Define binding energy, proximity, induced fit, orientation; relate to catalysis.
J. Use serine protease & HIV protease mechanisms to illustrate specificity, transition state, catalytic strategies.

Page 9 – Catalysis Fundamentals

• Catalyst purpose: Speeds attainment of equilibrium by lowering $\Delta G^{\ddagger}$.
• Transition state (TS): Peak along reaction‐coordinate diagram; enzyme binds TS better than substrate.
• Enzyme vs. non‐biological catalyst:
  • Remarkable rate enhancement (10⁶–10¹⁷), specificity, regulation, conditions (≈pH 7, 37 °C).
• Thermodynamics: $\Delta G^o$, $\Delta H$, $\Delta S$ unchanged → equilibrium position (Keq) unaffected.
• Lowering barrier in both forward & reverse directions.
• Temperature effect: Arrhenius equation $k=A e^{-\frac{E_a}{RT}}$; T increases molecular energy, raises fraction of molecules surpassing TS.

Page 10 – Enzymes in Disease & Pharmacology

• Loss-of-function mutations: Mis-sense altering key residues → deficient activity (Classic examples: PKU, G6PD deficiency).
• Gain/constitutive activity: Kinase oncogenes in cancer (e.g., BCR-ABL).
• Drug strategies:
  • Active‐site suicide inhibitors (aspirin acetylates Ser530 of COX-1).
  • Prodrugs dependent on metabolic enzymes for activation (e.g., clopidogrel → CYP2C19).
• Precision medicine: Genotyping metabolic enzymes predicts drug response.

Page 11 – Anatomy of the Active Site

• Binding/catalytic cleft: 3-D pocket often formed by residues distant in primary sequence.
• Induced fit: Binding energy from non-covalent contacts drives conformational change to TS complementarity.
• Catalytic residues: Common nucleophiles/acid-base: Ser, Cys, His, Lys, Glu, Asp.
• Cofactors: Metals (Zn²⁺, Mg²⁺) or organic (NAD⁺, FAD, PLP) extend chemistry.
• Energy landscape: More, stronger contacts with TS>substrate → preferential stabilization.

Page 12 – Specificity & Selectivity Principles

• Lock-and-key vs. induced fit: Real enzymes discriminate by stereochemistry; e.g., L-amino acid oxidase ignores D-isomer.
• Stereospecific outcome: Enzymatic hydration of fumarate → only L-malate.
• Dependence on tertiary structure: Denaturation abolishes specificity.
• pH sensitivity: Ionization of catalytic residues or substrate charges modulates binding/chemistry.

Page 13 – Quantitative Factors Affecting Rate

1. [S] dependence: Hyperbolic curve described by $v=\frac{V<em>{\max}[S]}{K</em>M+[S]}$; saturation at $V_{\max}$.
2. Temperature: Optimal ≈37 °C for human enzymes; beyond, denaturation reduces rate.
3. pH profile: Bell-shaped; optimum reflects required protonation of key residues (see Page 14).

• Clinical note: Pepsin optimum ≈pH 2 (stomach), alkaline phosphatase optimum ≈pH 9.

Page 14 – Molecular Basis of pH Effects

1. Catalytic residue ionization: Example lysozyme – Glu35 must be protonated, Asp52 deprotonated for acid-base catalysis.
2. Additional reasons:
   • Substrate charge complementarity;
   • Global protein stability (ionic bonds, salt bridges);
   • Metal cofactor coordination (pH alters ligands).

• Buffering capacity of physiological fluids maintains enzymes near optimal pH, yet microenvironments vary (lysosome vs. cytosol).

Page 15 – Enzyme Classification & Nomenclature

1. Net reaction diversity parallels organic chemistry: redox, group transfer, hydrolysis, etc., but enzymes accelerate and direct them.
2. Naming: root = substrate + “-ase” or reaction; EC number denotes class–subclass–sub-subcategory–serial.
   – Example: Lactate dehydrogenase = EC 1.1.1.27 (oxidoreductase).

• Clinical relevance: Plasma enzyme panel uses tissue-specific isoforms (CK-MB, LDH-1) to diagnose injury.

Page 16 – Energy Coordinate Diagram Basics

• Plot: Reaction progress (x) vs. free energy (y).
• Activation energy $E<em>a$ determines rate: $k \propto e^{-E</em>a/RT}$.
• Transition state: apex; fleeting, high-energy, non-isolable.
• Thermodynamic driving force: $\Delta G^o$ between S and P.
• Enzymes lower $E_a$ but leave $\Delta G^o$ unchanged.

Page 17 – Quantitative Relation ΔG° and Keq

$\Delta G^o = -RT\ln K_{eq}$.
• Lowering $\Delta G^{\ddagger}$ (activation) affects rate constant _k_, not equilibrium constant _K_.
• Thus catalysts do not shift chemical equilibria.

Page 18 – Source of Activation Energy Reduction

• Weak binding interactions (H-bonds, ionic, van der Waals) release binding energy (ΔG_b) used to offset activation barrier.
• Mechanisms enumerated on next page.

Page 19 – Four Major Catalytic Principles

1. Binding Energy (ΔG_b): Preferential TS binding lowers barrier.
2. Proximity (Effective Molarity): Bringing substrates into same micro‐volume enhances collision frequency.
3. Induced Fit: Conformational changes position catalytic groups & strain substrates toward TS.
4. Orientation: Active site aligns reactive atoms with proper orbital overlap.

• Figure 8.4 illustrates complementarity shifts from substrate to TS.

Page 20 – Lock-and-Key Model Critique

• Deficiency: Explains specificity but not rate acceleration; stabilizes substrate more than TS → would raise, not lower, barrier.
• Modern view: Dynamic induced fit + conformational selection.

Page 21 – Induced Fit Evidence

• Kinetic isotope effects, X-ray structures of enzyme bound to TS analogs (e.g., lysozyme + NAM-Streptomycin) show distortions.
• Conformational changes observed in hexokinase, HIV protease, kinases via crystallography.

Page 22 – Induced Fit in Hexokinase

• Problem: ATP is abundant; without conformational gating, enzyme would waste ATP on water.
• Mechanism: Glucose binding triggers large domain closure, excluding water and aligning ATP γ-phosphate with C6-OH → specificity.
• Prevents futile hydrolysis ensuring metabolic economy.

Page 23 – Strategy 1: Covalent Catalysis

• Temporary covalent bond between enzyme nucleophile (often Ser, Cys, Lys, His) and substrate.
• Forms acyl-enzyme or Schiff base; later hydrolyzed to regenerate enzyme.
• Example: Ser195 attack in chymotrypsin.

Page 24 – Strategy 2: Acid–Base Catalysis (Chymotrypsin)

• General acid/base: His57 alternately accepts and donates protons to facilitate nucleophilic attack and leaving-group departure.
• Concert with covalent catalysis – catalytic triad (Ser195–His57–Asp102).
• Reaction: Peptide bond cleavage generating tetrahedral intermediate stabilized by oxyanion hole.

Page 25 – Strategy 3: Metal Ion Catalysis

• Example: Zn²⁺ in carbonic anhydrase polarizes water → OH⁻; also stabilizes negative charge on bicarbonate product.
• Roles: Charge shielding, redox cycling (Fe²⁺/Fe³⁺), substrate orientation.
• Mnemonic: ‘CHOPS’ – Carbonic anhydrase, Histone deacetylase, Oxygenases, Polymerases, Superoxide dismutase.

Page 26 – Strategy 4: Cofactor Catalysis

• LDH + NAD⁺/NADH: Hydride transfer between C = O of pyruvate & nicotinamide ring.
• Cofactor provides chemistry (electron sink, carriers) unavailable to amino acids alone.
• Regulation: Cellular [NADH]/[NAD⁺] ratio affects pathway flux (fermentation vs. respiration).

Page 27 – Serine Protease Family Overview

• Physiological roles: Digestion (trypsin, chymotrypsin, elastase), coagulation (thrombin), fibrinolysis (plasmin), immunity (complement C1), developmental (cocoonase).
• Clinical relevance: Dysregulation in emphysema (elastase), thrombosis, complement deficiencies.
• Shared catalytic triad but differing specificity pockets.

Page 28 – Substrate Specificity Determinants

• Scissile bond preference upstream residue (P1 position):
  • Chymotrypsin: bulky hydrophobics (Phe, Trp, Tyr).
  • Elastase: small Ala, Gly (due to pocket with Thr & Val narrowing).
  • Trypsin: basic Arg, Lys (Asp189 at bottom of pocket).
• ‘Serine protease’ designation from active nucleophile Ser195.
• Structural composition: β-barrel fold, disulfide network ensures stability in harsh environments (GI tract).

Page 29 – Chymotrypsin Catalytic Mechanism (Stepwise)

1. Substrate binding: Aromatic side chain inserts into hydrophobic pocket; positions peptide bond next to Ser195.
2. Nucleophilic attack: Ser195-O⁻ (activated by His57, stabilized by Asp102) attacks carbonyl → tetrahedral intermediate stabilized by oxyanion hole (Gly193, Ser195 amide NH).
3. Acyl-enzyme formation: Collapse releases C-terminal peptide fragment, enzyme now acylated.
4. Water activation: His57 deprotonates water; OH⁻ attacks acyl carbonyl → second tetrahedral intermediate.
5. Product release & regeneration: Intermediate collapses, releasing N-terminal fragment; Ser195 restored.

Page 30 – Transition State Stabilization Emphasis

• Oxyanion hole: Donor NH groups donate H-bonds to developing negative charge; lowers $\Delta G^{\ddagger}$.
• Energetic advantage: Binding energy of TS analogs (e.g., peptide boronic acids) extremely high (nM) confirming complementarity.

Page 31 – Hydrolytic Deacylation (Water as Nucleophile)

• Concept: Enzyme not merely scaffold but actively alters water pKₐ via His‐mediated proton abstraction, converting weak nucleophile into strong hydroxide.
• Significance: Illustrates enzyme’s capacity to perform difficult chemistry under physiological conditions.

Page 32 – Catalytic Cycle Completion

• Key takeaways:
  • Enzyme emerges unaltered (true catalyst).
  • Rate enhancement ~10⁹-fold compared to uncatalyzed peptide hydrolysis (half-life years).
  • Mechanistic understanding guides inhibitor design (serine‐trap drugs, e.g., DFP).

Page 33 – Enzymes & HIV Life Cycle

• Targets along HIV replication: Reverse transcriptase (RT), integrase, protease.
• Pharmaceutical focus: Inhibitors mimicking TS of protease cleavage events (peptidomimetics).
• Multistep therapy: Combines entry/fusion blockers, RT inhibitors, protease inhibitors to limit resistance.

Page 34 – HIV Aspartyl Protease Mechanism vs. Chymotrypsin

• Catalytic dyad: Asp25 (both monomers) – uses activated water similar to serine proteases but no covalent acyl intermediate.
• TS: Tetrahedral hemiketal collapses → cleavage.
• Design implication: Hydroxyethylamine isosteres mimic TS geometry and resist further hydrolysis.

Page 35 – Inhibitor‐Based Pharmaceutical Example

• Compound: 1,2-epoxy-3-(4-nitrophenoxy)propane – irreversible aspartic protease inactivator.
• Mechanism: Covalent modification of Asp; time-dependent & pH-dependent (one Asp protonated).
• Broader principle: Suicide inhibitors achieve selectivity via enzyme’s own catalysis; can be pH-tuned.

Page 36 – Closing Slide / Course Reference

• Series title: “Cell Bio & Physio | Enzymes as Catalysts 1.”
• Indicates continuation into subsequent lectures (Catalysts 2, Kinetics, Regulation).
• Integration: Knowledge here underpins pharmacology, metabolism, and disease modules.