Unit 3: Proteins and Enzymes

Proteins are linear polymers of L-α-amino acids joined by peptide bonds, folding into defined three-dimensional shapes that determine biological function; enzymes are the catalytic subset of proteins. This unit builds on the chemistry of the amino acid and the peptide bond, so those properties are fixed first.

I. Orientation: The Amino Acid and the Peptide Bond

The behaviour of every protein traces back to 20 standard amino acids and the geometry of the bond linking them.

• Monomer structure: a central α-carbon bearing an amino group (–NH₃⁺), a carboxyl group (–COO⁻), a hydrogen, and a variable R-group (side chain) that defines identity.
• Side-chain classes: nonpolar (Ala, Val, Leu, Ile, Phe), polar uncharged (Ser, Thr, Asn, Gln), acidic (Asp, Glu), basic (Lys, Arg, His); this polarity dictates folding and catalysis.
• Peptide bond: an amide bond formed by condensation between the –COOH of one residue and –NH₂ of the next, releasing H₂O.
• Partial double-bond character: resonance of the C–N bond makes the peptide unit planar and rigid; the six atoms Cα–C(=O)–N(H)–Cα lie in one plane.
• Rotatable bonds: only the bonds flanking the α-carbon rotate — φ (phi, N–Cα) and ψ (psi, Cα–C) — the degrees of freedom that all backbone conformation depends on.
• Levels of structure: primary (sequence), secondary (local H-bonded folds), tertiary (whole-chain fold), quaternary (subunit assembly).

II. Proteins

Architecture, roles, and physical stability

Protein structure is hierarchical; classification and function both follow from how the chain folds and what its surface presents.

A. Structural classification of proteins

Proteins are grouped by overall shape and solubility, which correlate with role.

• Fibrous proteins: elongated, insoluble, repetitive secondary structure serving support and protection.
  • α-keratin: coiled-coil of α-helices in hair and nail; stabilised by disulfide (–S–S–) cross-links.
  • Collagen: triple helix of Gly-X-Y repeats (X often Pro, Y often hydroxyproline); every third residue is glycine because only H fits the crowded core.
• Globular proteins: compact, roughly spherical, water-soluble, hydrophobic core buried inside.
  • Examples: myoglobin, haemoglobin, most enzymes; act in transport, catalysis, regulation.
• Membrane proteins: hydrophobic surfaces span the lipid bilayer as transmembrane α-helices or β-barrels; e.g. transporters and receptors.
• Classification by secondary-structure content (folds):
  • all-α: e.g. myoglobin; all-β: e.g. immunoglobulin domains; α/β and α+β: mixed, e.g. TIM barrel.

B. Function of proteins

Function follows the folded surface and any bound cofactor; the same chemistry recurs across roles.

• Catalysis: enzymes lower activation energy (e.g. carbonic anhydrase, ~10⁶ reactions s⁻¹).
• Transport and storage: haemoglobin carries O₂ via a bound heme Fe²⁺; ferritin stores iron.
• Structural support: collagen in tendon, elastin in ligament.
• Movement: actin and myosin generate force in muscle contraction.
• Signalling and regulation: hormones (insulin) and receptors transmit information.
• Defence: immunoglobulins bind antigens with specific complementarity.
• Regulation of gene expression: transcription factors bind DNA through motifs such as the helix-turn-helix.

C. Ramachandran plot

The Ramachandran plot maps which backbone conformations are sterically allowed by plotting φ against ψ (Ramachandran, 1963).

ψ (deg)  +180 ┌───────────────┐
              │  β-sheet      │
           0  │        αR     │
              │               │
        -180  └───────────────┘
              -180    φ    +180

• Axes: x = φ (rotation about N–Cα), y = ψ (rotation about Cα–C), each spanning −180° to +180°.
• Allowed regions: clusters where atoms do not clash sterically.
  • Right-handed α-helix (αR): φ ≈ −57°, ψ ≈ −47°.
  • β-sheet: φ ≈ −120°, ψ ≈ +120°.
  • Left-handed α-helix: small allowed patch, φ ≈ +57°, ψ ≈ +47°; rare, usually glycine.
• Residue exceptions:
  • Glycine: no side chain, so it samples much of the plot — extra flexibility.
  • Proline: ring locks φ near −60°, restricting conformation.
• Use: validates a solved structure — residues falling in disallowed regions flag modelling errors.

D. Stability of proteins and denaturation

The native fold is only marginally stable, held by many weak interactions whose loss unfolds the chain.

• Stabilising forces:
  • Hydrophobic effect: burial of nonpolar side chains away from water; the dominant contribution.
  • Hydrogen bonds: backbone C=O···H–N define α-helix and β-sheet.
  • Ionic (salt) bridges: between oppositely charged side chains, e.g. Asp⁻···Lys⁺.
  • Disulfide bonds: covalent –S–S– links between cysteines, locking tertiary structure.
• Marginal net stability: typical ΔG of folding is only ~20–60 kJ mol⁻¹, the small difference between large opposing enthalpic and entropic terms.
• Denaturation: loss of native 3-D structure (and function) without breaking peptide bonds.
  • Heat: disrupts H-bonds and hydrophobic packing (egg albumin coagulating).
  • pH extremes: alter side-chain charge, breaking salt bridges.
  • Chaotropes: urea, guanidinium chloride solvate exposed groups.
  • Detergents/organic solvents: disrupt the hydrophobic core.
  • Reducing agents: β-mercaptoethanol cleaves –S–S– bonds.
• Reversibility: ribonuclease refolds spontaneously on removal of urea (Anfinsen), showing sequence encodes the fold.

III. Enzymes

Biological catalysts: naming, rates, and mechanism

Enzymes are proteins (a few are RNA) that accelerate reactions by binding substrate at an active site without being consumed.

A. Classification of enzymes

Enzymes are named systematically by the reaction they catalyse, each assigned an EC number (EC class.subclass.sub-subclass.serial).

• EC 1 Oxidoreductases: transfer electrons; e.g. lactate dehydrogenase (oxidation using NAD⁺).
• EC 2 Transferases: move a functional group between molecules; e.g. hexokinase transfers phosphate.
• EC 3 Hydrolases: cleave bonds by adding water; e.g. trypsin, lipase.
• EC 4 Lyases: add/remove groups to form or break double bonds without hydrolysis; e.g. pyruvate decarboxylase.
• EC 5 Isomerases: rearrange atoms within a molecule; e.g. phosphoglucose isomerase.
• EC 6 Ligases: join two molecules using ATP; e.g. DNA ligase.
• Cofactors: many require metal ions (Zn²⁺, Mg²⁺) or organic coenzymes (NAD⁺, FAD, coenzyme A) for activity.

B. Overview of enzyme kinetics

Kinetics describes how reaction velocity depends on substrate concentration, formalised by the Michaelis–Menten model.

• Reaction scheme: enzyme (E) binds substrate (S) to form complex (ES), then releases product (P).

E + S ⇌ ES → E + P
       k1,k-1   k2

• Michaelis–Menten equation:

v = (Vmax · [S]) / (Km + [S])

• v: initial reaction velocity.
• Vmax: maximal velocity when enzyme is saturated.
• [S]: substrate concentration.
• Km: Michaelis constant, [S] at which v = Vmax/2; = (k₋₁ + k₂)/k₁.
  • Meaning of Km: low Km signals high substrate affinity; it is independent of enzyme amount.
  • kcat and efficiency: kcat = Vmax/[E]ₜ is the turnover number; kcat/Km measures catalytic efficiency (limit ~10⁸–10⁹ M⁻¹ s⁻¹).
  • Lineweaver–Burk linearisation: plotting 1/v vs 1/[S] gives a straight line, slope Km/Vmax, y-intercept 1/Vmax.

1/v = (Km/Vmax)(1/[S]) + 1/Vmax

• Worked point: if Vmax = 100 µmol min⁻¹ and Km = 2 mM, then at [S] = 2 mM, v = (100 × 2)/(2 + 2) = 50 µmol min⁻¹, exactly half Vmax.

C. Factors affecting activity of enzymes

Activity responds to conditions that alter enzyme structure, substrate availability, or the active site.

• Substrate concentration: v rises with [S] then plateaus at Vmax as sites saturate (hyperbolic curve).
• Enzyme concentration: v is directly proportional to [E] when substrate is in excess.
• Temperature: rate rises with heat until the optimum (~37 °C for human enzymes), then falls sharply as denaturation sets in.
• pH: each enzyme has an optimum (pepsin ~2, trypsin ~8); extremes change ionisation of catalytic residues.
• Inhibitors:
  1. Competitive: resembles substrate, binds active site; raises apparent Km, Vmax unchanged; overcome by more substrate (e.g. malonate on succinate dehydrogenase).
  2. Non-competitive: binds elsewhere, lowering effective enzyme; Vmax falls, Km unchanged.
• Activators and cofactors: metal ions or coenzymes are required for a functional active site.

D. Mechanism of enzyme catalysis

Enzymes accelerate reactions by stabilising the transition state, lowering activation energy (Ea) rather than changing ΔG.

• Substrate binding models:
  1. Lock-and-key: active site is pre-shaped to fit the substrate exactly (Fischer).
  2. Induced fit: binding reshapes the active site around the substrate (Koshland), the more accurate picture.
• Catalytic strategies:
  • Transition-state stabilisation: the site binds the strained transition state most tightly, cutting Ea.
  • Acid–base catalysis: side chains donate or accept protons; e.g. His in the catalytic triad.
  • Covalent catalysis: a transient covalent enzyme–substrate intermediate forms; e.g. Ser in chymotrypsin.
  • Metal-ion catalysis: bound metal orients substrate or stabilises charge; e.g. Zn²⁺ in carboxypeptidase.
  • Proximity and orientation: binding aligns reactive groups, raising effective concentration.
• Worked case — serine protease triad: Ser–His–Asp cooperate; His deprotonates Ser, whose oxygen attacks the peptide carbonyl to form a covalent acyl-enzyme, then water hydrolyses it, releasing product and regenerating the enzyme.