Mode of action of enzymes

1. Overview

Enzymes are globular proteins that act as biological catalysts: they speed up reactions in living organisms while remaining unchanged themselves.

Each enzyme has a small region called the active site, formed by the folding of its polypeptide chain. The shape of the active site is complementary to one particular substrate. When the substrate binds, it forms a temporary enzyme-substrate complex, and this lowers the activation energy so the reaction proceeds faster.

The precise fit between substrate and active site is why enzymes are specific. Two models, the lock-and-key and induced-fit hypotheses, describe how binding happens. This topic also covers how the progress of enzyme-catalysed reactions is followed in the laboratory, including the use of a colorimeter.

Key Definitions

• Enzyme: a globular protein that acts as a biological catalyst, speeding up a specific reaction without being used up or permanently changed.
• Intracellular enzyme: an enzyme that catalyses a reaction inside the cell that made it, such as catalase.
• Extracellular enzyme: an enzyme secreted by a cell to catalyse a reaction outside the cell, such as amylase.
• Active site: the region of an enzyme with a specific three-dimensional shape that is complementary to its substrate, where the substrate binds.
• Enzyme-substrate complex: the temporary structure formed when a substrate binds to the active site of an enzyme.
• Activation energy: the minimum amount of energy that reacting molecules must have for a reaction to start.
• Enzyme specificity: the property that an enzyme usually catalyses only one reaction because its active site is complementary to only one type of substrate.
• Lock-and-key hypothesis: the model that the substrate fits a rigid active site of exactly the right shape, like a key fitting a lock.
• Induced-fit hypothesis: the model that the active site is flexible and changes shape slightly as the substrate binds, moulding closely around it.

Content

Enzymes are globular proteins

Every enzyme is a protein with a specific tertiary structure: its polypeptide chain folds into a compact, roughly spherical (globular) shape, held together by:

• hydrogen bonds
• ionic bonds
• disulfide bonds
• hydrophobic interactions

This folding brings particular amino acids together to form the active site. Because the active site's shape depends on the precise tertiary structure, anything that disrupts that structure (such as high temperature or extreme pH) changes the active site and stops the enzyme working.

How enzymes lower activation energy

Enzymes work by providing an alternative reaction pathway with a lower activation energy. They do not:

• change the overall energy of the reactants or products
• change whether a reaction can happen

By lowering the activation energy, a much greater proportion of molecules have enough energy to react at any given temperature, so the reaction goes faster. As catalysts, enzymes are not used up, so a single enzyme molecule converts substrate after substrate.

Intracellular and extracellular enzymes

Enzymes are grouped by where they act.

• Intracellular enzymes catalyse reactions inside the cell that made them. A classic example is catalase, which breaks down the toxic by-product hydrogen peroxide into water and oxygen.
• Extracellular enzymes are made inside a cell but secreted to work outside it. For example, amylase is secreted into the gut, where it catalyses the hydrolysis of starch into maltose. The maltose is then further hydrolysed by maltase into glucose, which is small enough to be absorbed.

Secreting digestive enzymes lets large food molecules be broken down outside cells before the smaller products are taken up.

Forming the enzyme-substrate complex

The substrate binds to the active site to form an enzyme-substrate complex. While bound, the substrate is held in a position and orientation that makes the reaction far more likely: bonds in the substrate are strained or brought close to reactive groups, which lowers the activation energy. The products then leave the active site, freeing the enzyme to bind another substrate molecule.

Because the active site is complementary to only one type of substrate, each enzyme normally catalyses only one reaction. A substrate of the wrong shape cannot fit, so no enzyme-substrate complex forms. This is why enzymes are described as specific.

Two models of binding: lock-and-key vs induced-fit

Two hypotheses describe how the substrate fits the active site. Both explain specificity; the induced-fit model is the more accurate.

Feature	Lock-and-key hypothesis	Induced-fit hypothesis
Active site shape	Rigid and already exactly right	Flexible
What happens on binding	Substrate simply slots in	Active site changes shape slightly to mould around the substrate
Effect on the substrate	Held in place	Bonds strained further, lowering activation energy more
Everyday analogy	A key fitting a lock	A hand changing shape to grip an object
Explains specificity?	Yes, but oversimplified	Yes, only a roughly correct substrate induces the right change

Following the progress of a reaction

The course of an enzyme-catalysed reaction can be followed either by measuring how fast product appears or by measuring how fast substrate disappears.

Feature	Rate of product formation	Rate of substrate disappearance
Enzyme used	Catalase	Amylase
Substrate	Hydrogen peroxide	Starch
What is measured	Oxygen gas released	Starch remaining
How it is measured	Volume of oxygen collected (e.g. in a gas syringe) at set time intervals	Samples tested at intervals with iodine in potassium iodide solution
What you observe	Volume of oxygen rises, then levels off	Blue-black colour fades towards orange-brown as starch is broken down

In both methods the reaction is fastest at the start, when substrate concentration is highest, and slows as substrate is used up, giving a curve that levels off into a plateau. The graph below illustrates this shape: the amount of product rises steeply at first and then flattens once almost all the substrate has been converted.

Be careful which graph you are explaining. On this product-against-time graph the plateau means the substrate has been used up. This is not the same as the plateau on a rate-against-substrate-concentration graph, which levels off because the active sites are saturated and the enzyme concentration has become the limiting factor (this is the $V_{max}$ plateau, covered in topic 3.2). Do not write "substrate used up" for a saturation plateau, or "active sites saturated" for a time-course plateau.

When interpreting these graphs, track the empty active sites. The proportion of empty active sites is highest at the very start (before substrate has bound) and again at the very end (when the reaction is complete and substrate is exhausted), and lowest in between when most active sites are occupied.

Worked example

Exam-style question: A student adds the enzyme catalase to hydrogen peroxide and collects the oxygen released in a gas syringe, recording the volume every 10 seconds. The graph of oxygen volume against time rises steeply at first and then gradually levels off. Explain the shape of this graph. [3]

Model answer:

• At the start the substrate (hydrogen peroxide) concentration is highest, so there are many successful collisions with active sites and many enzyme-substrate complexes form per second, giving the fastest rate (steepest gradient).
• As the reaction proceeds the substrate is used up, so fewer complexes form per second and the rate of oxygen release decreases (the gradient becomes shallower).
• The line levels off when almost all the substrate has been broken down, so no more oxygen can be produced.

Worked example

Exam-style question: Two reactions, A and B, were followed by collecting the oxygen released by catalase. The volume of oxygen produced by reaction A reached 16 cm³ after 40 s, with the curve clearly tangent to a straight line through the origin for the first 20 s, over which 12 cm³ was released. Reaction B was identical except that the substrate concentration was halved. (a) Calculate the initial rate of reaction A. (b) State and explain how the initial rate of reaction B would compare with that of A. [4]

Model answer:

• (a) The initial rate is found from the gradient of a tangent drawn at $t = 0$. Over the first 20 s the curve follows a straight line through the origin, so the initial rate $= \dfrac{12\ \text{cm}^3}{20\ \text{s}} = 0.6\ \text{cm}^3\,\text{s}^{-1}$.
• (b) The initial rate of reaction B would be lower (roughly half, about $0.3\ \text{cm}^3\,\text{s}^{-1}$).
• With half the substrate concentration, there are fewer substrate molecules per unit volume, so fewer successful collisions with active sites and fewer enzyme-substrate complexes form per second at the start, giving a less steep tangent at $t = 0$.

Using a colorimeter

When a reaction involves a colour change, a colorimeter gives a more reliable, quantitative measurement than judging colour by eye. How it works:

• Light is passed through the sample to a sensor, which measures absorbance (how much light is absorbed) or transmission (how much passes through).
• A suitable coloured filter is chosen so the colour being measured is absorbed strongly.
• The colorimeter is first set to zero using a blank.
• A calibration curve of absorbance against known concentrations is produced so unknown samples can be read off.

For example, in the amylase/iodine method the blue-black colour fades as starch is digested. By first making a calibration curve of absorbance against known starch concentrations, you can read the starch concentration off the curve from each sample's absorbance. This turns the qualitative colour fade into actual concentration values: plotting starch concentration against time then gives the rate of substrate disappearance numerically rather than by eye.

Key Equations

This topic is mainly qualitative, so there are no set formulae to learn. The one quantitative skill is finding the rate of reaction from a graph. The initial rate is the gradient of the tangent drawn at the start:

$$\text{initial rate} = \frac{\Delta(\text{amount of product})}{\Delta(\text{time})} \quad \text{at } t = 0$$

For example, an oxygen volume of $12\ \text{cm}^3$ released in the first $20\ \text{s}$ gives an initial rate of $0.6\ \text{cm}^3\,\text{s}^{-1}$.

Common Mistakes to Avoid

• Saying that amylase produces a product "small enough to be absorbed". Amylase hydrolyses starch to maltose, but maltose is a disaccharide and is not absorbed directly; it must first be hydrolysed by maltase into glucose before it can be absorbed.
• Misreading "empty active site" graphs. The proportion of empty active sites is highest at the start (no substrate has bound yet) and highest again at the end (the reaction is complete and the substrate is used up), not steadily falling throughout.
• Thinking enzyme-substrate complexes only form at the very beginning. As long as there is still a reasonable amount of substrate present, complexes are continually forming and breaking down, so their concentration stays roughly constant during the middle of the reaction. The concentration only falls once the substrate becomes scarce near the end.
• Saying an enzyme "speeds up a reaction by adding energy" or "raises the energy of the products". An enzyme lowers the activation energy by providing an alternative pathway; it does not change the overall energy change of the reaction.
• Confusing the two hypotheses. The lock-and-key model treats the active site as rigid; the induced-fit model says the active site is flexible and changes shape as the substrate binds. Do not describe the lock-and-key active site as changing shape.
• Saying hydrolysis "releases water". Hydrolysis reactions, such as the breakdown of starch, use water to split molecules; it is condensation reactions that release water.
• Writing that the enzyme is "used up" as a reaction slows. Enzymes are catalysts and are reused; a reaction slows because the substrate is being used up, not the enzyme.

Exam Tips

• Use the word complementary (not "same shape" or "identical") to describe how the substrate fits the active site.
• Always name the active site and enzyme-substrate complex when explaining how enzymes work; vague answers about "the enzyme joining the substrate" lose marks.
• When asked about lowering activation energy, add that the enzyme provides an alternative pathway and does not change the overall energy change.
• To find an initial rate, draw a tangent at $t = 0$ and calculate its gradient; remember the units (e.g. $\text{cm}^3\,\text{s}^{-1}$).
• For graph questions, describe the trend and then explain it in terms of substrate concentration, successful collisions and enzyme-substrate complexes.
• For a compare question (e.g. lock-and-key vs induced-fit), make linked points using comparative words such as "whereas", "both" and "unlike" rather than describing each model in turn; aim to give at least one similarity and one difference.
• When choosing a method to follow a reaction, link product formation to catalase/oxygen and substrate disappearance to amylase/iodine, and mention a colorimeter for any colour change so the result is quantitative rather than judged by eye.