Enzymes — A-Level Biology Revision Notes

How Enzymes Work

Enzymes are globular proteins with a specific three-dimensional shape determined by their tertiary structure. Each enzyme has a region called the active site — a small cleft or pocket with a shape that is complementary to its specific substrate. Reactions that break down large molecules into smaller ones (breaking bonds) are called catabolic reactions. Reactions that build up larger molecules from smaller ones (forming bonds) are called anabolic reactions. Together, all of an organism’s catabolic and anabolic reactions make up its metabolism.

The Lock and Key Hypothesis

This model proposes that the active site of the enzyme has a shape that is perfectly complementary to the substrate — like a key fitting into a lock. The substrate enters the active site to form an enzyme-substrate complex (ESC). The reaction occurs, products are formed, and because the products are no longer complementary in shape to the active site, they leave. The enzyme is unchanged and can be reused. This model explains enzyme specificity (only one substrate fits) and enzyme recovery, but it does not explain how the activation energy is lowered. It also implies a rigid active site, which makes it harder to explain how the substrate enters.

The Induced Fit Theory

This more advanced model explains what the lock and key hypothesis cannot. In this model, the active site is not initially 100% complementary to the substrate — it is approximately complementary, which makes it easier for the substrate to enter. Once the substrate binds, the active site moulds around the substrate, changing shape to form a precise fit. This conformational change distorts and strains the bonds within the substrate, weakening them so that the activation energy needed to break them is significantly reduced. This allows reactions to proceed at the relatively low temperatures found in living organisms. Lysozyme and hexokinase are two enzymes known to work by the induced fit mechanism.

Activation Energy

On an energy profile diagram, the activation energy is shown as the “hump” that must be overcome for reactants (substrates) to be converted into products. Without an enzyme, this hump is large — meaning high temperatures or extreme conditions would be needed. With an enzyme, the hump is significantly smaller because the induced fit mechanism weakens the bonds in the substrate. The products have lower energy than the substrate because energy (heat) is released during the formation of the products.

Calculating the Rate of Reaction

Rate is not something you measure directly — it must be calculated. Rate is the change in a measurable quantity over a given time period: Rate = Change ÷ Time. The measurable quantity (dependent variable) might be the volume of oxygen produced, the number of bubbles, the distance moved by a bubble, or the mass of product formed.

From a graph where time is on the x-axis, rate is calculated as the gradient of the line using the equation: gradient = (y₂ − y₁) ÷ (x₂ − x₁). For a straight-line graph, the gradient can be read directly. For a curved line, you must draw a tangent to the curve at the point of interest and then calculate the gradient of the tangent.

Practical Methods for Measuring Rate

Common methods include: timing how long a paper disc takes to float to the surface of a liquid (gas production makes it buoyant), counting bubbles per unit time, measuring the volume of water displaced by a gas using an inverted measuring cylinder or burette, and using a gas syringe to directly measure gas volume.

Percentage Error

Percentage error = (uncertainty ÷ quantity measured) × 100. When using a graduated scale (burette, measuring cylinder), the uncertainty is ±0.5 of the smallest division. When two measurements are combined (e.g. measuring enzyme volume then substrate volume), the uncertainties are added together. To reduce percentage error: use instruments with smaller graduations, or increase the quantity measured.

The Effect of Enzyme Concentration

As enzyme concentration increases, the rate of reaction increases because there are more active sites available, so more enzyme-substrate complexes (ESCs) form per unit time and more product is produced. This relationship is directly proportional in the initial region of the graph.

Eventually, the rate reaches a plateau (maximum rate) and becomes constant. At this point, substrate concentration becomes the limiting factor — most of the substrate has been converted to product, so many enzyme molecules are without substrate and are not forming ESCs. Increasing the enzyme concentration further has no effect because there is insufficient substrate to occupy all the active sites.

The Effect of Substrate Concentration

As substrate concentration increases, the rate increases because more substrate molecules are available to collide with enzyme active sites, forming more ESCs and producing more product. Again, this is directly proportional initially.

At high substrate concentrations, the rate plateaus. Enzyme concentration is now the limiting factor. All active sites are continuously occupied by substrate molecules — the enzymes are saturated and working at maximum capacity. Adding more substrate cannot increase the rate because there are no free active sites.

The Effect of Temperature

Temperature has two distinct effects on enzyme activity, producing a characteristic asymmetric bell curve:

Below the optimum temperature: As temperature increases, the enzyme and substrate molecules gain kinetic energy and move faster. This causes more frequent collisions between enzyme and substrate, forming more ESCs and producing more product. The rate increases up to the optimum temperature — the temperature at which the enzyme works at its maximum rate.

Above the optimum temperature: The enzyme molecules vibrate so vigorously that hydrogen bonds (and other weak bonds maintaining the tertiary structure) begin to break. This causes a change in the tertiary structure of the enzyme, altering the shape of the active site so that it is no longer complementary to the substrate. No ESCs can form, no product is produced, and the rate falls to zero. This process is called denaturation and it is permanent — the enzyme cannot recover its original shape.

The Effect of pH

Each enzyme has a specific optimum pH at which it works at its maximum rate. The optimum pH varies between enzymes — for example, pepsin (stomach) has an optimum pH of about 2, while amylase (mouth/intestines) has an optimum of about 7–9.

Small changes in pH away from the optimum affect the charges on key amino acids in the active site. The active site contains amino acids with charged R-groups (positive or negative) that interact with complementary charges on the substrate to form the ESC. If the pH changes, the concentration of H⁺ or OH⁻ ions alters these charges, preventing the substrate from binding. This reduces the rate but does not permanently damage the enzyme.

Large changes in pH break ionic bonds and hydrogen bonds that maintain the tertiary structure, causing denaturation — a permanent change in the shape of the active site. No ESCs can form and the rate falls to zero.

Competitive Inhibition

A competitive inhibitor has a similar shape to the normal substrate of the enzyme. Because of this similarity, the competitive inhibitor can enter the active site and form an enzyme-competitive-inhibitor complex, physically blocking the substrate from binding.

Crucially, the binding of a competitive inhibitor is reversible — the inhibitor can leave the active site unchanged, freeing the enzyme to bind with the normal substrate. At any given moment, the substrate and the competitive inhibitor are competing for the same active site.

The Effect of Substrate Concentration

At low substrate concentrations: The competitive inhibitor occupies many active sites because it faces little competition. The rate is significantly reduced compared to the uninhibited reaction.

At high substrate concentrations: Substrate molecules outnumber the competitive inhibitor and outcompete it for active sites. The enzyme eventually reaches its maximum rate — the same V_max as the uninhibited reaction. The competitive inhibitor is effectively swamped.

A named example: malonate is a competitive inhibitor of succinate dehydrogenase (which converts succinate to fumarate).

Non-Competitive Inhibition

A non-competitive inhibitor has a different shape from the substrate and does not bind to the active site. Instead, it binds to an allosteric site — a separate region on the enzyme. This binding causes a conformational change in the enzyme that alters the shape of the active site, so the substrate can no longer form an ESC. The enzyme is effectively denatured.

The Effect of Inhibitor Concentration

At low inhibitor concentrations: Only a few enzymes are denatured. The remaining functional enzymes still form ESCs, but the rate can never reach the maximum because some enzymes are permanently non-functional. The rate plateaus at a lower level than the uninhibited reaction.

At high inhibitor concentrations: Most enzymes are denatured. The rate is severely reduced and plateaus at a very low level.

Increasing substrate concentration cannot overcome non-competitive inhibition — the inhibitor does not compete for the active site, so adding more substrate has no effect on the inhibited enzymes. The V_max is always reduced.

Examples include heavy metal ions (mercury, silver, arsenic) that bind permanently to sulfhydryl (-SH) groups, and cyanide, which irreversibly binds to ferric iron (Fe³⁺) in cytochrome oxidase, blocking aerobic respiration.

End-Product Inhibition

A metabolic pathway is a series of linked reactions in which the product of one reaction becomes the substrate for the next, with each step catalysed by a different enzyme: A → B → C (catalysed by E₁ then E₂).

In end-product inhibition (also called allosteric control), the final product of a metabolic pathway acts as a non-competitive inhibitor of the first enzyme in the pathway. When enough of the end product has accumulated, it binds to the allosteric site of E₁, changing its shape and preventing it from catalysing the first reaction. This switches off the entire pathway. When the end product is used up and its concentration falls, the allosteric site is freed, E₁ regains its functional shape, and the pathway restarts.

This is a form of negative feedback that regulates metabolism — it prevents the overproduction of end products and conserves raw materials and energy. A named example is the metabolism of the amino acid threonine, where the end product isoleucine inhibits the first enzyme (threonine deaminase) in the pathway.

Immobilised Enzymes

Methods of Immobilisation

In practical work, enzymes are commonly immobilised by entrapment in alginate beads. The enzyme solution is mixed with sodium alginate, and droplets are added to calcium chloride solution. The calcium ions cause the alginate to gel, trapping the enzyme inside small beads.

Immobilised vs Free Enzymes

Immobilised enzymes have a higher optimum temperature and denature at higher temperatures than free enzymes because the inert matrix stabilises the tertiary structure, protecting the hydrogen bonds from breaking. However, enzymes trapped inside beads (entrapment) may have a slower rate than surface-immobilised enzymes because the substrate must diffuse into the bead to reach the active site, whereas enzymes on cellulose fibres are fully exposed.

Biosensors — Medical Applications

Biosensors use immobilised enzymes to detect and measure the concentration of specific biological molecules (e.g. glucose in blood or urine). There are two main types:

Qualitative Biosensors (Clinistix)

Clinistix are test strips with two immobilised enzymes (glucose oxidase and peroxidase) plus a colourless dye, all fixed onto an absorbent pad. When dipped in urine containing glucose: glucose oxidase converts glucose to gluconic acid and hydrogen peroxide → peroxidase uses the hydrogen peroxide to convert the colourless dye to a coloured product. The intensity of the colour is compared to a standard colour chart. This gives a qualitative result — no numerical value is generated, but the approximate glucose concentration range can be determined. The colour intensity is directly proportional to the glucose concentration.

Quantitative (Digital) Biosensors

A digital biosensor produces a precise numerical reading. Its components work in sequence:

Advantages of digital biosensors: they are specific (enzyme specificity), sensitive (detect very low concentrations), rapid (results in minutes), require only a small sample, and produce quantitative (numerical) results.

Industrial Applications — Lactose-Free Milk

Immobilised enzymes are widely used in continuous production processes. A key example is the production of lactose-free milk for people with lactose intolerance — a condition where the person cannot produce the enzyme lactase, resulting in undigested lactose being fermented by gut bacteria (causing abdominal pain and diarrhoea).

A column is packed with immobilised lactase (entrapped in alginate beads). Milk is fed in at the top and flows down through the column. The lactase hydrolyses the lactose into glucose and galactose. Lactose-free milk exits at the bottom.

Factors Affecting Product Yield

Flow rate: A faster flow rate means the milk passes through the column more quickly, reducing contact time between substrate and enzyme. Fewer ESCs form, so less lactose is hydrolysed and the product may be contaminated with remaining lactose. A slower flow rate increases contact time and yield.

Bead size: Smaller beads pack more closely together, creating a larger total surface area for enzyme-substrate interaction. The closer packing also increases resistance to flow, effectively slowing the flow rate and further increasing contact time. Both effects increase product yield.

Temperature: Higher temperatures increase kinetic energy, forming more ESCs and increasing yield — but the temperature must remain below the enzyme’s denaturation point.

Advantages of Immobilised Enzymes in Industry

The enzyme does not contaminate the product. It can be easily recovered and reused, reducing costs (enzymes are expensive). Multiple enzymes can be used simultaneously. Enzymes can be easily added or removed from the process. Immobilised enzymes tolerate more extreme conditions (higher temperatures, wider pH range) because the matrix stabilises their structure.

Where Enzymes Appear on Your Specification

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