Enzymes

Enzymes are proteins, and are biological catalysts. They can speed up the rate of biological reactions (e.g. respiration, photosynthesis) without being used up themselves.

Why is this important? Most essential life processes (e.g. digestion, respiration) would happen far too slowly for us to be alive without enzymes!

Enzymes can work in two main places:

• Intracellular: Enzymes that are present within the cells (i.e. inside the cell membrane). Examples could include:
  • DNA polymerase, responsible for building new DNA strands and present within the nucleus (inside the cell)
  • RNA polymerase, used in transcription to create mRNA
  • Catalase, responsible for breaking down hydrogen peroxide inside cells
• Extracellular: Enzymes that are released to work outside cells. Examples can include:
  • Digestive enzymes (e.g. amylase, which is in your saliva, or proteases, which are present in the small intestine). These are present outside the cells in your gut, allowing nutrients to be broken down efficiently and absorbed

Activation Energy

So how do enzymes (these biological catalysts) actually speed up the reactions?

They work by lowering the reaction’s activation energy. This is the amount of energy required for the reaction to get started.

A reaction needs energy (activation energy) to get started, as it needs to break the existing chemical bonds in the reactant molecules.

If you look at the graph below, can you see that the reaction catalysed by an enzyme needs a lower activation energy than the uncatalysed reaction?

Why couldn’t we just use heat as a supply of energy? Why do we have to use enzymes? In our bodies, our cells work at around 37 °C, which is nowhere near hot enough for most reactions to get going on their own. Similarly, other living organisms have temperature limits. Note: in industry, you’ll notice they often supply heat and catalysts to speed up the rate of reactions.

Enzyme Specificity & Active Site

Can any enzyme then catalyse any reaction? No, an enzyme catalyses one reaction, or one type of reaction. This is called specificity, and it comes directly from the enzyme’s shape: its active site.

Enzymes have an ‘active site’, which is specific to certain substrates.

If you remember back to the proteins topic, enzymes are proteins, and a protein’s structure determines its function.

Remember back to protein structure…

1. The sequence of amino acids in the polypeptide (its primary structure) is determined by a gene
2. That sequence determines how the chain folds, and which bonds form between R groups, giving the enzyme its tertiary structure
3. The tertiary structure determines the precise 3D shape of the active site
4. Only a substrate with a shape complementary to the active site can bind and form an enzyme-substrate complex

So if a substrate isn’t complementary to the active site, it cannot bind, and the enzyme cannot catalyse that reaction. This is why a change to the amino acid sequence (through a gene mutation) can stop an enzyme functioning (if it changes the tertiary structure of the active site).

The Induced-Fit Model

So we know that enzymes are specific (have a specific active site for certain substrates), and they lower the activation energy of reactions, but how do they actually do this?

This is where the most recent induced-fit model comes in.

It was originally explained by the lock and key model (you probably remember it from GCSE), which says the active site was a fixed shape and that the substrate fitted in exactly, like a lock and key.

This does help to explain the specificity part well, but it needed to be refined to take into account that the enzyme’s shape changed slightly once it bound to the specific substrate (i.e. it didn’t stay completely fixed). So the lock and key model was refined to the induced-fit model.

What is the induced-fit model?

1. The substrate is not a perfect fit for the active site to begin with
2. As the substrate binds, the enzyme’s active site changes shape slightly. This moulds closely around the substrate, and results in a tighter fit
3. This tighter fit puts strain on the bonds within the substrate
4. The strained bonds break more easily, which lowers the activation energy
5. Products are formed and released, and the active site returns to its original shape.

Factors Affecting Rate of Reaction

Let’s start by defining what rate of reaction means. This can be looked at in two ways:

• how quickly substrate is used up, or
• how quickly product is formed

Now, let’s look at the factors that affect the rate of reaction:

• Enzyme concentration
• Substrate concentration
• Inhibitors (competitive or non-competitive)
• pH
• Temperature

Enzyme Concentration

A higher enzyme concentration means more active sites available, so more enzyme-substrate complexes can form at any one time, and the rate increases.

• As enzyme concentration increases, rate increases proportionally, as long as there is plenty of substrate

Substrate Concentration

A higher substrate concentration means there will be more successful collisions with active sites, so more enzyme-substrate complexes form and the rate increases.

• As substrate concentration increases, rate increases at first

Competitive Inhibitors

A competitive inhibitor has a shape similar to the substrate, so it is complementary to the active site.

• It binds to the active site itself
• While it is bound, the substrate cannot enter, so fewer enzyme-substrate complexes form and the rate falls
• The substrate and inhibitor are effectively competing for the same active sites

This means that…increasing the substrate concentration reduces the effect of the inhibitor. With more substrate molecules around, they are more likely to reach an active site before an inhibitor does. So the maximum rate can still be reached, it just takes a higher substrate concentration to get there.

Non-Competitive Inhibitors

A non-competitive inhibitor binds somewhere other than the active site.

1. The inhibitor binds to a site other than the active site (the allosteric site)
2. This changes the enzyme’s tertiary structure
3. Because the tertiary structure has changed, the active site changes shape
4. The substrate is no longer complementary to the active site, so it cannot bind and fewer enzyme-substrate complexes form

This means that…increasing substrate concentration does not overcome a non-competitive inhibitor, because the substrate and inhibitor aren’t competing for the same site. The enzyme has changed shape so cannot bind to the substrate, so the rate of reaction will decrease.

pH

Every enzyme has an optimum pH at which its rate of reaction is highest. Move away from that optimum in either direction and the rate falls.

Why does pH have this effect?

• A change in pH means a change in the concentration of hydrogen ions
• These interfere with the ionic bonds and hydrogen bonds that hold the enzyme’s tertiary structure in place

• As those bonds break, the active site changes shape and is no longer complementary to the substrate
• Fewer enzyme-substrate complexes form, so the rate drops. At extreme pH the enzyme becomes denatured (this means the active site changes shape so much that the substrate no longer fits)

Temperature

Similarly to pH, every enzyme has an optimum temperature, where the rate of reaction is the highest, but above the optimum the enzyme becomes denatured.

Below the optimum

• Increasing temperature gives molecules more kinetic energy
• Substrate and enzyme molecules move faster, so there are more frequent collisions between substrates and active sites
• More enzyme-substrate complexes form per second, so the rate increases

Above the optimum

• The extra energy makes the enzyme molecule vibrate more
• This vibration breaks the hydrogen bonds and ionic bonds holding the tertiary structure together
• The active site changes shape and is no longer complementary to the substrate
• The enzyme is denatured, and unlike the effect of cooling, this is permanent

Required Practical 1

The variable you investigate (the independent variable) is usually temperature, pH, substrate concentration or enzyme concentration, i.e. one of the factors we explored above that can change the rate of reaction

Measuring the rate

These are common scenarios:

• Catalase + hydrogen peroxide: collect the oxygen gas produced and measure its volume over time
  • How it works: catalase breaks hydrogen peroxide down into water and oxygen. The faster the enzyme (catalase) works, the more oxygen is collected each minute (e.g. in a gas syringe or an upturned measuring cylinder)
• Amylase + starch: sample at intervals and test with iodine solution, timing how long until it no longer goes blue-black (all starch broken down)
  • How it works: amylase hydrolyses starch into maltose, and iodine only turns blue-black when starch is present, so once it all turns orange-brown, all the starch has been broken down
• Trypsin + milk: time how long the cloudy milk takes to turn clear as the protein is hydrolysed
  • How it works: milk is cloudy because of the protein casein in milk. The enzyme trypsin hydrolyses it into smaller soluble polypeptides, so the milk goes clear. The faster it clears, the higher the rate of reaction.

In exams, you’ll usually be given an unfamiliar enzyme and reaction (e.g. polyphenol oxidase browning apple tissue, or lactase). Focus on the principles below: how rate is measured, which variables to control, and what makes a valid control.

Calculating rate

There are two methods, depending on what you measured.

Method 1: 1 ÷ time taken (when you only know how long the reaction took)

• Rate = 1 ÷ time taken
• Example: at 30 °C, the starch is all broken down after 40 s (we know this because the iodine changes colour from blue-black to orange-brown)
  • So rate = 1 ÷ 40 = 0.025 s⁻¹
  • If instead we measured it at 40 °C, we find it only takes 25 s
    • So rate = 1 ÷ 25 = 0.04 s⁻¹. A shorter time means a faster rate of reaction.

Method 2: the gradient of a graph (when you measured the amount of product or substrate over time)

• Use the initial rate: the rate at the very start of the reaction, where the curve is steepest. Why? As the reaction goes on, substrate is used up, so the rate slows down. If you measured later, you’d be seeing the effect of the substrate running out, not the effect of the variable you’re testing.
• To find the initial rate, draw a tangent:
  1. Draw a straight line (a tangent) that just touches the curve at time 0, following the steepest part at the start
  2. Pick a point on the tangent and draw a large triangle down to the time axis (the bigger the triangle, the more accurate your reading)
  3. Initial rate = gradient of the tangent = change in y ÷ change in x, with units (e.g. cm³ s⁻¹)
• Read the values off the tangent, not the curve: the curve has already started to slow down

Controlling the other variables

Whichever variable you are testing, the others must be kept constant:

• Keep temperature constant with a water bath
• Keep pH constant with a buffer solution
• Keep volumes and concentrations of enzyme and substrate the same
• Leave time for solutions to reach the set temperature before mixing

Exam Question Practice