Mode of Action of Enzymes

Almost every reaction in a living cell would be far too slow to keep the cell alive if it were not catalysed by an enzyme. Enzymes are proteins whose precise shape lets them bind one particular substrate and speed up its reaction without being used up. This note explains how they do it: the active site, the enzyme–substrate complex, the two hypotheses of how substrate and enzyme fit together, and the lowering of activation energy. It also covers how to follow the progress of an enzyme-catalysed reaction in the laboratory, which is examined in Paper 3 and in data questions on Paper 2.

Enzymes are globular proteins

Because an enzyme is a globular protein, everything in Proteins applies to it. Its polypeptide chain (or chains) is folded into a precise tertiary structure held by hydrogen bonds, ionic bonds, disulfide bonds and hydrophobic interactions. Hydrophilic R groups on the outside make it soluble, so it can work in the watery cytoplasm or in body fluids.

Enzymes work in two places:

• Intracellular enzymes catalyse reactions inside cells. Examples: catalase, which breaks down toxic hydrogen peroxide in liver and many other cells; DNA polymerase in the nucleus; the enzymes of respiration in the cytoplasm and mitochondria; ATP synthase on the inner mitochondrial membrane.
• Extracellular enzymes are made inside cells, secreted (by exocytosis) and catalyse reactions outside the cells that made them. Examples: amylase in saliva and pancreatic juice, which hydrolyses starch to maltose; trypsin and lipase in the small intestine; enzymes secreted by fungi to digest their food externally.

Extracellular enzymes are made on ribosomes on the rough endoplasmic reticulum and packaged by the Golgi body into secretory vesicles (see Eukaryotic cells).

The active site and specificity

Only a small part of an enzyme takes part in catalysis. This region, usually a cleft or pocket on the surface of the molecule, is the active site.

The active site is formed by the folding of the polypeptide, so its shape depends directly on the enzyme's tertiary structure, which in turn depends on its primary structure (amino acid sequence). Amino acids that are far apart in the primary structure can lie side by side in the active site after folding.

Because the active site has a specific shape, each enzyme can bind only one substrate, or a small group of substrates with very similar shapes. This is enzyme specificity. For example, amylase hydrolyses the α-1,4 glycosidic bonds in starch but cannot hydrolyse the β-1,4 bonds in cellulose, even though both are glycosidic bonds between glucose molecules: the shape of the substrate is different.

The enzyme–substrate complex

Catalysis happens in a sequence of steps:

1. The enzyme and substrate molecules move about randomly and collide.
2. If the substrate collides with the active site in the right orientation, it binds to the active site, held by temporary bonds (hydrogen bonds and ionic interactions) with R groups in the active site. This forms an enzyme–substrate complex (ES complex).
3. The reaction takes place: bonds in the substrate are broken (or new bonds formed between two substrates), forming an enzyme–product complex (EP complex).
4. The products have a different shape from the substrate, so they no longer fit the active site and are released.
5. The enzyme is unchanged and its active site is free to bind another substrate molecule.

Because the enzyme is released unchanged, a small number of enzyme molecules can catalyse the conversion of a very large number of substrate molecules. The number of substrate molecules converted per second by one enzyme molecule is called the turnover number; for catalase it is about 40 million per second, one of the highest known.

Lock-and-key and induced-fit hypotheses

Two models explain how the substrate fits the active site. You must be able to describe both and say why the second is now preferred.

The lock-and-key hypothesis

The lock-and-key hypothesis says that the active site has a fixed, rigid shape that is exactly complementary to the substrate, just as only one key fits a lock. The substrate (key) fits the active site (lock) precisely, forming an ES complex.

This explains specificity well, but it treats the enzyme as rigid. Evidence shows that enzyme molecules are flexible and that the active site changes shape when the substrate binds.

The induced-fit hypothesis

The induced-fit hypothesis says that the active site is not exactly complementary to the substrate before binding. When the substrate enters, the active site (and sometimes the whole enzyme) changes shape slightly to mould itself around the substrate, so that it fits more closely. The substrate "induces" the fit.

This change of shape:

• brings catalytic R groups in the active site into exactly the right positions;
• puts strain on bonds in the substrate, distorting them so they break more easily;
• explains why some molecules that resemble the substrate can bind but are not converted (the active site does not change shape correctly around them);
• explains how one enzyme can act on a small range of related substrates.

Feature	Lock-and-key	Induced fit
Shape of active site before binding	rigid; exactly complementary to substrate	flexible; roughly complementary to substrate
What happens when substrate binds	substrate slots in; no change in enzyme shape	active site changes shape to fit substrate more closely
How bonds in substrate are weakened	not explained well	change of shape distorts/strains bonds in substrate
Explains specificity?	yes	yes
Explains a small range of substrates?	poorly	yes
Current status	simple model; useful starting point	supported by evidence; preferred model

Lowering the activation energy

For any reaction to happen, the reacting molecules must gain a minimum amount of energy to break or weaken existing bonds. This energy barrier is the activation energy. In the body, at around $37\ ^\circ\text{C}$, very few molecules have enough kinetic energy to overcome a high activation energy, so uncatalysed reactions are extremely slow.

(Horizontal axis: progress of reaction; vertical axis: energy. The reactants start at an energy level of 6 and the products end at 3. The taller curve is the uncatalysed reaction, with an activation energy shown by the arrow from the reactant level to the top of the barrier. The lower curve is the same reaction catalysed by an enzyme: the barrier is much lower, so the activation energy is smaller. The overall energy change, reactants to products, is the same in both.)

The enzyme lowers the activation energy because, in the ES complex:

• two substrate molecules are held close together in the correct orientation, so they react more easily than if they had to meet by chance with enough energy;
• the change in shape on binding (induced fit) strains bonds in the substrate, making them easier to break;
• charged or polar R groups in the active site create a microenvironment that favours the reaction, for example by donating or accepting $\text{H}^+$.

The result is that, at the same temperature, a far greater proportion of molecules can react, so the rate increases enormously. The enzyme does not change the overall energy change of the reaction, and it does not change the products formed.

Following the progress of an enzyme-catalysed reaction

The rate of an enzyme-catalysed reaction can be measured in two ways:

• the rate of formation of a product, for example oxygen from hydrogen peroxide (catalase);
• the rate of disappearance of a substrate, for example starch hydrolysed by amylase.

Either way, you get a progress curve: the amount of product (or substrate) against time.

(Horizontal axis: time / s; vertical axis: volume of oxygen collected / cm³. The curve is steepest at the start and levels off at 40 cm³ when all the hydrogen peroxide has been broken down. The line through the origin is the tangent at $t = 0$, whose gradient is the initial rate. The other straight line is the tangent at $t = 30\ \text{s}$, which is less steep.)

The shape of the progress curve has an explanation that examiners ask for repeatedly:

1. At the start the substrate concentration is highest. There are many collisions between substrate and active sites, so many ES complexes form per unit time and the rate is fastest. The curve is steepest.
2. As the reaction proceeds substrate is used up, so its concentration falls. There are fewer successful collisions and fewer ES complexes per unit time, so the rate falls. The gradient decreases.
3. The curve levels off (plateaus) when all the substrate has been used up. No more product forms. The enzyme is still active: it simply has nothing left to act on.

The final amount of product depends only on the amount of substrate at the start, not on the amount of enzyme.

Catalase: measuring product formation

Catalase catalyses the breakdown of hydrogen peroxide, a toxic by-product of metabolism:

$$2\text{H}_2\text{O}_2 \longrightarrow 2\text{H}_2\text{O} + \text{O}_2$$

The oxygen can be measured by:

• collecting it in a gas syringe, or over water in an inverted measuring cylinder, and recording the volume at regular intervals;
• counting bubbles per minute (less precise: bubbles vary in size);
• the floating disc method: filter-paper discs soaked in catalase sink in hydrogen peroxide solution, then rise as oxygen bubbles form on them. The time for a disc to rise is measured and $\text{rate} = 1/t$.

Sources of catalase include potato, liver, celery and yeast suspension.

Amylase: measuring substrate disappearance

Amylase hydrolyses starch to maltose. Starch gives a blue-black colour with iodine in potassium iodide solution, and the colour disappears as starch is hydrolysed. The progress of the reaction can be followed by:

• Sampling onto a spotting tile: at regular intervals (e.g. every 30 s), a drop of the reaction mixture is added to a drop of iodine solution on a white tile. The time at which the drop no longer turns blue-black (it stays orange-brown) is the time when all the starch has gone, called the achromic point. Rate $= 1/t$.
• Using a colorimeter to measure the decrease in the intensity of the blue-black colour.

Using a colorimeter

A colorimeter measures how much light of a chosen wavelength is absorbed by a coloured solution. The more concentrated the coloured substance, the higher the absorbance (and the lower the transmission). This turns a subjective colour judgement into a number.

Colorimeters are also used with Benedict's solution (measuring the remaining blue colour, or the colour of the filtrate), and with other reactions that produce or remove a colour.

Practice questions

Answers

1. A globular protein that acts as a biological catalyst, increasing the rate of a (metabolic) reaction without being permanently changed or used up.
2. Intracellular enzymes act inside the cell that made them, e.g. catalase (or DNA polymerase, respiratory enzymes). Extracellular enzymes are secreted and act outside the cell that made them, e.g. amylase (or trypsin, lipase).
3. The active site is a small region (cleft/pocket) of the enzyme, formed by a few amino acids, where the substrate binds and the reaction is catalysed; its shape is determined by the enzyme's tertiary structure (folding of the polypeptide, held by bonds between R groups); it has a shape complementary to only one substrate (or a few closely related substrates), so only that substrate can bind and form an enzyme–substrate complex.
4. The active site is not exactly complementary to the substrate before binding; when the substrate binds, the active site (enzyme) changes shape slightly to fit more closely around the substrate; this puts strain on bonds in the substrate / holds substrates in the correct orientation, so activation energy is lowered; on release of products the active site returns to its original shape.
5. Axes: energy (vertical) and progress of reaction (horizontal). Reactants at a higher level, products at a lower level (for an energy-releasing reaction). Two humps: a tall one labelled "without enzyme" and a lower one labelled "with enzyme". Activation energy shown as the vertical distance from reactants to the top of each hump; the one with enzyme is smaller. Overall energy change is the same for both.
6. Rate $= 1/12.5 = 0.080\ \text{s}^{-1}$.
7. At the start, substrate concentration is known and the same in all experiments (none has yet been used up) and no product has built up; later the rate falls as substrate is used up by different amounts in each experiment, so the mean rate does not reflect the conditions being tested.
8. Rate $= (33.4 - 31.1)/10 = 0.23\ \text{cm}^3\ \text{s}^{-1}$. Between 0 and 10 s it was $10.4/10 = 1.04\ \text{cm}^3\ \text{s}^{-1}$. It is lower because much of the hydrogen peroxide has been broken down, so the substrate concentration is lower, there are fewer collisions with active sites and fewer ES complexes form per second.
9. Absorbance starts high because starch–iodine is dark blue-black; it decreases over time as starch is hydrolysed to maltose (which does not give a blue-black colour with iodine), so less light is absorbed; the decrease is fastest at first (highest starch concentration, most ES complexes) and slows; absorbance levels off at (or near) the baseline value when all the starch is hydrolysed.
10. Any six: the active site of lysozyme is (roughly) complementary to the polysaccharide in bacterial cell walls; the substrate binds to the active site forming an enzyme–substrate complex; held by hydrogen bonds/ionic interactions with R groups of amino acids in the active site; the active site changes shape (moulds around) the substrate as it binds (induced fit); this distorts/strains the glycosidic bond, making it easier to break; R groups in the active site (e.g. charged groups) take part in the reaction / donate $\text{H}^+$; so activation energy is lowered. Starch has a different shape from the cell-wall polysaccharide (different monomers/bond arrangement), so it is not complementary to the active site; it cannot bind or does not induce the correct change in shape, so no ES complex forms and its bonds are not hydrolysed.