Proteins and enzymes

Amino Acids and Peptide Bonds

Amino acids are the monomers that build proteins, each sharing a common backbone but differing in their side chain (R group).

Every amino acid contains a central carbon bonded to four groups: an amine group (-NH₂), a carboxyl group (-COOH), a hydrogen atom, and a variable R group. The R group differs between the 20 amino acids found in living organisms. This variation gives each amino acid its unique chemical properties. Specific R group structures are not required for this specification.

Amino acids join together through condensation reactions to form peptide bonds. The amine group of one amino acid reacts with the carboxyl group of the next. This reaction releases one water molecule and forms a covalent peptide bond between the two amino acids. Two joined amino acids form a dipeptide, while many joined amino acids form a polypeptide. Proteins consist of one or more polypeptide chains folded into specific three-dimensional shapes.

Levels of Protein Structure

Proteins have four levels of structural organisation, each contributing to the final three-dimensional shape. The primary structure determines all higher levels because it dictates where bonds can form during folding.

Primary structure is the specific sequence of amino acids in a polypeptide chain. This sequence is determined by the gene encoding that protein. Amino acids in primary structure are joined only by peptide bonds.

Secondary structure forms when the polypeptide chain coils or folds into regular patterns. Hydrogen bonds form between the slightly negative oxygen of one peptide bond and the slightly positive hydrogen of another. Two main types occur:

• α-helix: the chain coils into a right-handed spiral.
• β-pleated sheet: the chain folds into flat, sheet-like structures.

Tertiary structure is the overall three-dimensional shape of a single polypeptide chain. The chain folds further as R groups interact with each other. Several bond types stabilise this structure:

Quaternary structure exists only in proteins made of two or more polypeptide chains. The chains assemble together, sometimes with non-protein prosthetic groups, to form the final functional protein.

The R groups dictate which bonds form during folding. So a single change in primary structure can alter the overall shape and disrupt protein function.

MisconceptionStudents often think that all four bond types form at every level of protein structure. Only peptide bonds form at primary level. Hydrogen bonds form at secondary level. The full range of bonds (hydrogen, ionic, disulfide, hydrophobic) appears only at tertiary and quaternary levels.

Exam TipMatch each bond type to the correct structural level.

Globular and Fibrous Proteins

Proteins fall into two main categories based on their shape and function. Globular proteins are spherical and water-soluble, while fibrous proteins are long, insoluble and structural.

Haemoglobin is a globular protein found in red blood cells that transports oxygen around the body. It has quaternary structure consisting of four polypeptide chains: two α-globin chains and two β-globin chains. Each chain contains a haem group [a prosthetic group containing an iron ion] that can bind one oxygen molecule. So one haemoglobin molecule transports up to four oxygen molecules. The hydrophilic R groups face outwards, making haemoglobin soluble in blood plasma and red blood cell cytoplasm. This solubility is essential for its transport role.

Collagen is a fibrous protein found in skin, bone, tendons, ligaments and cartilage. It consists of three polypeptide chains wound around each other in a triple helix, held together by hydrogen bonds. The chains are rich in the small amino acid glycine, allowing tight packing. Many collagen molecules cross-link by covalent bonds to form long, strong fibres. This structure gives collagen high tensile strength, which is ideal for its supportive and structural roles.

Enzyme Mechanism and Specificity

Enzymes are globular proteins that act as biological catalysts, speeding up biochemical reactions without being used up. Each enzyme has an active site with a three-dimensional shape complementary to its substrate.

Enzymes are highly specific because the active site only fits one substrate (or a small group of related substrates). This specificity arises from the enzyme’s tertiary structure, which itself depends on the primary sequence of amino acids. Because the active site shape is precisely defined, only the correct substrate can bind.

Two models describe how enzymes interact with substrates.

Lock and key model: The substrate fits exactly into a rigid active site, like a key into a lock. This model explains specificity but is now considered too simplistic.

Induced fit model (preferred): The active site is flexible. When the substrate first binds, the active site changes shape slightly to fit closely around the substrate. This conformational change strains bonds in the substrate, lowering the activation energy required for the reaction.

The full sequence of enzyme action is:

1. The substrate collides with the active site.
2. An enzyme-substrate complex forms.
3. The active site adjusts (induced fit), straining substrate bonds.
4. The reaction occurs and an enzyme-product complex forms briefly.
5. Products are released and the active site returns to its original shape.
6. The enzyme is unchanged and can catalyse another reaction.

Examiner InsightWhen asked to explain enzyme specificity, always link active site shape back to tertiary structure. Saying “the substrate fits the active site” is not enough on its own. State that the active site is complementary in shape to the substrate.

Exam TipUse “complementary,” not “same shape” or “identical.”

Enzymes and Activation Energy

All chemical reactions require activation energy [the minimum energy needed to start a reaction]. Without enzymes, this energy barrier is too high for most biological reactions to occur fast enough at body temperature.

Enzymes act as biological catalysts that lower the activation energy required. They achieve this by:

• Holding substrate molecules in the correct orientation for reaction.
• Straining bonds within the substrate (induced fit), making them easier to break.
• Providing a microenvironment in the active site that favours the reaction.

Because activation energy is reduced, reactions proceed much faster at the relatively low temperatures inside cells (around 37°C in mammals). Enzymes are not used up in the reaction. Once products are released, the enzyme is free to catalyse another reaction.

MisconceptionEnzymes do not “give energy” to reactions or “make reactions happen”. They simply lower the energy barrier so the reaction proceeds faster at lower temperatures. The reactants must still have sufficient kinetic energy to react.

Exam TipAlways say enzymes “lower activation energy”, never “provide energy”.

Intracellular and Extracellular Enzymes

Enzymes are classified by where they catalyse reactions in relation to the cell.

Intracellular enzymes catalyse reactions inside cells. They are produced by the cell and remain inside it. Examples include:

• Catalase: breaks down hydrogen peroxide (a toxic by-product of metabolism) into water and oxygen.
• DNA polymerase: catalyses DNA replication inside the nucleus.
• Respiratory enzymes inside mitochondria.

Extracellular enzymes are secreted by cells and catalyse reactions outside cells. They typically break down large molecules into smaller, absorbable units. Examples include:

• Amylase: secreted by salivary glands and pancreas; digests starch into maltose.
• Trypsin: a protease secreted by the pancreas that digests proteins in the small intestine.

The distinction matters because extracellular enzymes face changing conditions outside the cell. Intracellular enzymes work in the relatively stable environment of the cytoplasm or organelles.

Factors Affecting Enzyme Activity

Four key factors affect the rate of enzyme-catalysed reactions: temperature, pH, enzyme concentration, and substrate concentration.

Temperature: Increasing temperature raises the kinetic energy of molecules. Substrate-enzyme collisions become more frequent and successful. Rate increases until the optimum temperature is reached. Above this, the enzyme begins to denature [the tertiary structure breaks down as hydrogen and ionic bonds disrupt]. The active site loses its complementary shape, so substrates can no longer bind. Rate then falls sharply.

pH: Each enzyme has an optimum pH at which the active site shape is ideal for substrate binding. Above or below the optimum, hydrogen and ionic bonds in the tertiary structure disrupt, altering active site shape. Extreme pH values denature the enzyme. For example, pepsin’s optimum is around pH 2 (in the stomach), while trypsin’s is around pH 8 (in the small intestine).

Enzyme concentration: When substrate is in excess, increasing enzyme concentration increases rate proportionally because more active sites become available. If substrate becomes limiting, rate stops rising regardless of how much enzyme is added.

Substrate concentration: When enzyme concentration is fixed, increasing substrate concentration increases rate until all active sites are continuously occupied. At this point the rate plateaus at a maximum value ($V_{\mathrm{max}}$) because the enzyme becomes the limiting factor.

CORE PRACTICAL 4: Investigating Factors Affecting Initial Rate of Enzyme-Catalysed Reactions

Aim: To investigate how temperature, pH, enzyme concentration and substrate concentration each affect the initial rate of an enzyme-catalysed reaction.

Method

A common setup uses catalase (from yeast, potato, or liver) and hydrogen peroxide, measuring the volume of oxygen produced over time.

1. Place a known volume of hydrogen peroxide solution in a conical flask connected to a gas syringe.
2. Add a known mass or volume of catalase source and immediately start the timer.
3. Record the volume of oxygen produced every 10 seconds for 2 minutes.
4. Plot a graph of oxygen volume against time and calculate initial rate from the gradient of the steepest part (first 30 seconds).
5. Repeat the procedure at three to five different values of the chosen variable, keeping all others constant.
6. Plot a second graph of initial rate against the independent variable.

Variables

Independent variable (IV): The factor under investigation: temperature (°C), pH, enzyme concentration (g/cm³ or %), or substrate concentration (mol/dm³).

Dependent variable (DV): Initial rate of reaction (cm³ of oxygen produced per second).

Control variables (CV): All factors not under investigation; volume and concentration of solutions; same enzyme source; same apparatus.

Expected results

• Temperature: rate increases up to an optimum (around 40°C for catalase), then decreases sharply as the enzyme denatures.
• pH: a bell-shaped curve with peak rate at the optimum pH; extreme pH values denature the enzyme.
• Enzyme concentration: rate increases proportionally until substrate becomes limiting, then plateaus.
• Substrate concentration: rate increases steeply, then levels off at $V_{\mathrm{max}}$ as all active sites become saturated.

Precaution

Initial rate must be measured from the steepest part of the curve at the very start. As the reaction proceeds, substrate concentration falls and rate decreases. Measuring later gives an underestimate of the true initial rate. Wear eye protection because hydrogen peroxide is an irritant.