Topic 3: Enzymes
3.1 Mode of action of enzymes
Students should be able to:
1) state that enzymes are globular proteins that catalyse reactions inside cells (intracellular enzymes) or are secreted to catalyse reactions outside cells (extracellular enzymes)
2) explain the mode of action of enzymes in terms of an active site, enzyme–substrate complex, lowering of activation energy and enzyme specificity, including the lock-and-key hypothesis and the induced-fit hypothesis
3) investigate the progress of enzyme-catalysed reactions by measuring rates of formation of products using catalase and rates of disappearance of substrate using amylase
4) outline the use of a colorimeter for measuring the progress of enzyme-catalysed reactions that involve colour changes
1.1 Enzymes: definition and location
Enzymes are globular proteins that act as biological catalysts by speeding up chemical reactions without being consumed; they occur inside cells (intracellular enzymes such as DNA polymerase, catalase) where they catalyse metabolic pathways and are also secreted outside cells (extracellular enzymes such as amylase in saliva or pepsin in the stomach) to digest large molecules before absorption.
1.2 Mode of action: active site, ES complex, lowering activation energy, specificity; lock-and-key vs induced-fit
An enzyme’s catalytic function depends on its active site, a uniquely shaped pocket formed by the tertiary structure where the substrate binds to form an enzyme–substrate (ES) complex; binding positions substrates to lower the activation energy (by strain, proximity/orientation effects and microenvironment changes) thereby increasing reaction rate while the enzyme remains unchanged. Enzyme-substrate specificity is explained historically by the lock-and-key model (active site rigid and complementary to the substrate) and more modernly by the induced-fit model which states that substrate binding induces a conformational change in the enzyme that optimises interactions and catalytic efficiency — both models should be mentioned, with induced-fit preferred to explain how enzymes stabilise transition states and why specificity is high but not absolute.
1.3 & 1.4 Investigating enzyme-catalysed reactions: catalase, amylase and use of a colorimeter
To investigate enzyme activity you measure rates — for catalase measure the rate of formation of oxygen from hydrogen peroxide (for example by collecting gas over water or measuring displacement in a syringe) and calculate initial rate (volume O₂ time⁻¹) using a range of substrate concentrations or temperatures; for amylase measure the disappearance of starch (using iodine: record time for loss of blue-black colour) or the appearance of reducing sugars (Benedict’s) and calculate rate as change per unit time. When colour change is involved a colorimeter provides quantitative progress: set a wavelength that corresponds to the dye/colour produced (or the dye-substrate complex), zero the instrument with a blank, take absorbance readings at fixed intervals and convert absorbance to concentration using a calibration curve; initial rate is determined from the initial linear portion of concentration vs time. In all practicals control variables tightly, measure initial rates and repeat for reliability
3.2 Factors that affect enzyme action
Students should be able to:
1) investigate and explain the effects of the following factors on the rate of enzyme-catalysed reactions
2) explain that the maximum rate of reaction (Vmax ) is used to derive the Michaelis–Menten constant (Km ), which is used to compare the affinity of different enzymes for their substrates
3) explain the effects of reversible inhibitors, both competitive and non-competitive, on enzyme activity
4) investigate the difference in activity between an enzyme immobilised in alginate and the same enzyme free in solution, and state the advantages of using immobilised enzymes
2.1 Effects of factors on enzyme activity — temperature, pH, enzyme conc., substrate conc., inhibitor conc.
The rate of enzyme-catalysed reactions depends on several variables: temperature increases kinetic energy and collision frequency so rate rises with temperature until denaturation abruptly reduces activity at high temperatures; pH alters ionisation of amino acid residues at the active site so each enzyme has an optimum pH and activity falls either side due to disrupted bonding and shape change; enzyme concentration gives a directly proportional rise in initial rate if substrate is in excess (more active sites → more collisions) until other factors limit rate; substrate concentration increases rate hyperbolically: at low substrate the rate is approximately proportional to substrate, but as substrate rises enzymes become saturated and the rate approaches a maximum (Vₘₐₓ); inhibitor concentration generally reduces activity with the effect dependent on inhibitor type (see below) and increasing inhibitor concentration typically lowers initial rates. Experimentally vary only one factor at a time and measure initial rates to construct meaningful graphs.
2.2 Vmax and Km (Michaelis–Menten) — meaning and use
The maximum rate (Vₘₐₓ) is the asymptotic rate attained when all enzyme active sites are saturated with substrate; the Michaelis–Menten constant (Kₘ) is the substrate concentration at which the reaction rate is half of Vₘₐₓ and is used as an indicator of an enzyme’s affinity for its substrate (lower Kₘ means higher affinity under comparable conditions). Vₘₐₓ and Kₘ are derived from initial rate measurements across a range of substrate concentrations and fitted to the Michaelis–Menten equation or linearised plots (Lineweaver–Burk); Kₘ allows quantitative comparison of substrate affinity between enzymes or mutated variants.
2.3 Reversible inhibitors: competitive and non-competitive — effects on activity
Competitive reversible inhibitors resemble the substrate and compete for the active site, increasing the apparent Kₘ (lower apparent affinity) because more substrate is needed to reach half-Vₘₐₓ, but Vₘₐₓ remains unchanged because high substrate outcompetes the inhibitor. Non-competitive reversible inhibitors bind at an allosteric site different from the active site and reduce catalytic efficiency regardless of substrate concentration, lowering Vₘₐₓ while Kₘ remains essentially unchanged because substrate binding affinity is not directly affected. Both types reduce initial rates; the differences are best shown on graphs (Michaelis–Menten or Lineweaver–Burk) and stated numerically when possible. Also mention competitive inhibition can be overcome by increasing [S], whereas non-competitive cannot.
2.4 Immobilised enzymes in alginate vs free enzyme: differences, investigation and advantages
Immobilised enzymes are physically confined or localized (e.g., trapped inside calcium alginate beads) while retaining catalytic activity; compared with free enzymes they often show lower apparent activity per unit enzyme because of mass-transfer limitations (substrate must diffuse into the bead) and possible conformational constraints, but they offer practical advantages: easy separation of enzyme from products, enzyme reuse over many cycles, greater thermal and pH stability, suitability for continuous flow reactors and purer product streams. To investigate, run parallel assays under identical bulk conditions measuring initial rates for free enzyme and for enzyme immobilised in alginate beads (correcting for enzyme loading and accounting for diffusion delays), plot activity vs time or substrate concentration and comment on differences — expect slower initial apparent rates for immobilised enzyme but improved operational stability and reusability. Industrial and educational benefits (cost reduction, easy handling, use in biosensors) are important to mention.