01. Chapter 4 & 5

Protein Structure and Classification

  • Levels of Protein Structure

    • Primary Structure

    • Defined as the exact number and sequence of amino acids in a polypeptide chain.

    • Linked exclusively by covalent peptide bonds.

    • Information for primary structure is directed by DNA nucleotide sequences.

    • The primary sequence determines how the polypeptide chain folds into its final 3D conformation and dictates its specific biological function (e.g., lysozyme found in tears and saliva).


Primary structure of a polypeptide chain showing amino acids and peptide bonds

  • Secondary Structure

    • Regular coiling or folding of the repeating polypeptide backbone.

    • Maintained by hydrogen bonds formed between the oxygen (C=OC=O) and hydrogen (−NH-NH) atoms of the polypeptide backbone.

    • α\alpha-Helix: Coiled spiral shape (e.g., collagen fibers, keratin).

    • β\beta-Pleated Sheet: Parallel or anti-parallel pleated arrangement.


Secondary structure alpha helix and beta pleated sheet

  • Tertiary Structure

    • Precise, complex 3D bending and folding of the secondary structure into a compact globular or fibrous shape.

    • Maintained by interactions between variable amino acid R-groups:

      • Peptide Bonds: Covalent bonds along the main chain.

      • Hydrogen Bonds: Weakest interactions between polar R-groups.

      • Ionic Bonds: Stronger bonds formed between oppositely charged R-groups (e.g., −NH3+-NH_3^+ and −COO−-COO^-); sensitive to pH shifts.

      • Disulfide Bridges: Covalent, extremely strong bonds between sulfur-containing R-groups (cysteine amino acids).

      • Hydrophobic and Hydrophilic Interactions: Weak interactions where non-polar hydrophobic R-groups cluster internally away from water, while polar hydrophilic R-groups face outward toward aqueous environments.


Tertiary structure interactions including ionic, disulfide, hydrogen bonds and hydrophobic interactions

  • Quaternary Structure

    • Functional association of two or more individual polypeptide chains (subunits).

    • Maintained by the same covalent and non-covalent R-group interactions found in tertiary structure, but occurring between distinct polypeptide molecules.

    • Subunits can be identical (common in homomeric enzymes) or different (e.g., Insulin; Hemoglobin consisting of 4 subunits: 2 α\alpha-globin and 2 β\beta-globin chains).


Quaternary structure complex of protein molecules like hemoglobin

  • Globular Protein Features

    • Enzymes are specialized globular proteins.

    • Globular proteins possess a compact, spherical shape.

    • Highly soluble in water due to abundant hydrophilic R-groups positioned on the exterior surface.

Enzyme Action and Thermodynamics

  • Biological Function of Enzymes

    • Enzymes act as biological catalysts that accelerate metabolic reaction rates without being consumed or permanently altered.

    • Necessary because untolerated high temperatures would be required for reactions to occur fast enough to sustain life.

  • Mechanism of Lowering Activation Energy (EaE_a)

    • Activation energy (EaE_a) is the minimum energy needed for colliding molecules to undergo a chemical reaction.

    • Enzymes lower activation energy by offering an alternative pathway featuring a specialized 3D active site:

    • Anabolic / Synthesis Reactions: Binding two substrates holds them close together in correct spatial orientation, reducing electrostatic repulsion so bonds form easily.

    • Catabolic / Breakdown Reactions: Fitting the substrate into the active site places strain and physical stress on specific covalent bonds, destabilizing the substrate so it breaks up easily.


Activation energy graph of uncatalysed vs enzyme catalysed reaction

  • Models of Enzyme Action

    • Lock and Key Model

    • Active site has a rigid, pre-formed 3D shape complementary to the substrate.

    • Substrate collides and fits precisely into active site to form an enzyme-substrate complex (ESCESC).

    • Reactions occur, bonds are broken/formed, products leave the active site, and the enzyme remains structurally unaltered.


Lock and key model of enzyme action

  • Induced-Fit Hypothesis

    • Active site of the inactive enzyme is initially only partially complementary or non-complementary to the substrate.

    • Upon collision, entry of the substrate induces a conformational change (change in shape) in the active site/enzyme.

    • The active site moulds around the substrate to achieve a precise, complementary fit, forming an enzyme-substrate complex (ESCESC).

    • Catalytic amino acid residues move into exact alignment, placing strain on substrate bonds and lowering activation energy.

    • After reaction, a temporary product-enzyme complex is formed; products no longer fit the modified active site, are released, and the active site returns to its original conformation.


Induced fit model of enzyme action

Factors Affecting Enzyme Kinetics

  • Temperature

    • Increasing temperature elevates kinetic energy of enzymes and substrates, increasing collision frequency and successful ESCESC formation per second.

    • Rate increases up to optimum temperature.

    • Above optimum temperature, increased thermal vibrations break hydrogen and ionic bonds maintaining tertiary structure.

    • The specific 3D active site shape changes (denaturation); substrate can no longer fit or bind.

    • Protein denaturation above 50 ∘C50\,^\circ C is typically irreversible.

    • At low temperatures, enzymes do not denature; low kinetic energy simply reduces collision frequency.


Effect of temperature on enzyme activity

  • pH

    • Enzymes have an optimum pH where rate of reaction is maximum.

    • Changes in H+H^+ or OH−OH^- ion concentration interact with charged R-groups, disrupting ionic and hydrogen bonds holding the tertiary structure.

    • Active site shape is altered, preventing substrate binding; extreme pH causes denaturation.


Effect of pH on enzyme activity

  • Substrate Concentration

    • At low substrate concentrations, substrate is the limiting factor. Rate increases linearly with substrate concentration as more active sites are occupied per second.

    • At high substrate concentrations, the rate plateaus (Vmax⁡V_{\max}). All active sites are fully saturated/occupied; enzyme concentration becomes limiting.

  • Enzyme Concentration

    • At low enzyme concentrations, enzyme is limiting. Increasing enzyme concentration provides more available active sites, increasing rate linearly.

    • At high enzyme concentrations, rate plateaus because substrate concentration becomes limiting.

  • Initial Rate vs. Average Rate

    • Initial rate of reaction (t=0t = 0) is maximum rate because substrate concentration is at its highest, maximizing active site occupation.

    • As reaction proceeds, substrate depletes, collision frequency drops, and reaction rate slows.

    • Calculation formula for initial rate:

Rate=Volume of product collectedTime taken\text{Rate} = \frac{\text{Volume of product collected}}{\text{Time taken}}

  • Example: If 5.8 cm35.8\,\text{cm}^3 oxygen is collected in 30 seconds30\,\text{seconds} (0.5 min0.5\,\text{min}), rate is:

5.8 cm30.5 min=11.6 cm3 min−1\frac{5.8\,\text{cm}^3}{0.5\,\text{min}} = 11.6\,\text{cm}^3\,\text{min}^{-1}

Enzyme Inhibition and Regulation

  • Competitive Inhibition

    • Competitive inhibitor has a similar shape/structure to the substrate and is complementary to the active site.

    • Competes with substrate for binding to active site, forming enzyme-inhibitor complexes and blocking ESCESC formation.

    • Does not undergo catalysis.

    • Can be overcome by increasing substrate concentration; maximum