Enzymes

What enzymes are and why life depends on them

An enzyme is a protein that acts as a biological catalyst. A catalyst is a substance that increases the rate of a chemical reaction and is not changed by the reaction. Because it is not used up, a very small amount of enzyme can catalyse the conversion of a huge amount of substrate.

Enzymes are involved in all metabolic reactions. Metabolism is the sum of every chemical reaction happening in an organism, from respiration to protein synthesis, and almost every one of those reactions needs its own enzyme to run at a usable speed.

The reason enzymes matter is speed. Left to themselves, many of the reactions that keep a cell alive would take place far too slowly, and an organism could not survive at that rate. Enzymes lower the energy needed for a reaction to start, so it happens fast enough to sustain life.

• Enzymes are proteins, so they are affected by temperature and pH.
• They are catalysts, so they are not changed or used up by the reaction they speed up.
• They work inside cells and outside them, for example the digestive enzymes in the gut.
• Each enzyme acts on one particular substrate, which is why so many different enzymes exist.

The lock-and-key model: active site and enzyme-substrate complex

Enzymes are globular proteins. Part of the molecule folds to form a hollow called the active site. The shape of the active site is complementary to the shape of the substrate, so the two fit together exactly, like a key fitting its lock.

The substrate binds to the active site to form an enzyme-substrate complex. While the substrate is held in place the reaction takes place, and the substrate is converted into products.

The products have a different shape from the substrate, so they no longer fit the active site and are released. The enzyme is left exactly as it was, free to bind the next substrate molecule. This is why enzymes are described as reusable catalysts.

• Substrate = the molecule that the enzyme acts on.
• Active site = the part of the enzyme with a shape complementary to the substrate.
• Enzyme-substrate complex = the temporary structure formed when the substrate binds to the active site.
• Products = the new molecules released once the reaction is complete.

Specificity: why one enzyme will not do another's job

Specificity means that each enzyme catalyses only one reaction, or a very small group of similar reactions. This follows directly from the lock-and-key model: because the active site has one particular shape, only a substrate with a complementary shape can bind to it.

If the shape of the molecule is different, it will not fit the active site. No enzyme-substrate complex forms, and no reaction is catalysed. The enzyme is a lock that opens only one key.

Catalase is a good example: it breaks down hydrogen peroxide into water and oxygen, but it has no effect on other molecules. Amylase digests starch but has no effect on protein, because protein molecules do not fit its active site.

The effect of temperature on enzyme activity

As temperature rises, enzyme and substrate molecules gain kinetic energy and move faster. They collide more often and with more energy, so there are more effective collisions and the rate of reaction increases. The temperature at which the enzyme works fastest is its optimum temperature. For most human enzymes this is about 37 °C.

Above the optimum, the rate falls sharply. The bonds that hold the protein's folded shape vibrate so strongly that they break, and the active site loses its shape. The enzyme is now denatured. The substrate no longer fits the active site, no enzyme-substrate complexes can form, and the reaction stops.

Denaturation is permanent. Cooling the enzyme down again will not restore the shape of the active site. This is very different from the slowing that happens below the optimum, which is fully reversible on warming.

The effect of pH on enzyme activity

The bonds that hold an enzyme's folded shape are sensitive to hydrogen ion concentration, so changing the pH changes the shape of the molecule. Each enzyme has an optimum pH at which its active site is exactly the right shape for its substrate.

Away from the optimum pH, the active site changes shape, so the substrate fits less well and the rate of reaction falls. Far enough from the optimum, the enzyme becomes denatured and the reaction stops permanently.

Different enzymes have very different optima because they work in different places. Pepsin, a protease in the stomach, has an optimum of about pH 2 and needs acid conditions. The proteases of the small intestine have an optimum of about pH 8 and need alkaline conditions.

Enzyme concentration, substrate concentration and the rate of reaction

Enzyme concentration: adding more enzyme means there are more active sites available. More enzyme-substrate complexes form, so the rate of reaction increases. This only works while substrate is plentiful - once every substrate molecule is being acted on, adding more enzyme makes no difference.

Substrate concentration: at low substrate concentration most active sites are empty, so increasing the substrate increases the rate, because collisions between enzyme and substrate become more frequent. As concentration rises further, more active sites are occupied at any one moment, until all of them are working at full capacity. This is saturation, and the rate then levels off into a plateau.

The key idea is the limiting factor: the rate is controlled by whichever factor runs out first. When the curve flattens, the enzyme is no longer the limiting factor - the substrate is in excess and the enzyme is working as fast as it can.

• More enzyme means more active sites, so a faster rate, up to a plateau.
• More substrate means more frequent collisions, so a faster rate, up to a plateau.
• At saturation every active site is occupied, so adding more substrate cannot raise the rate.
• On a graph of rate against substrate concentration, the curve rises steeply and then flattens.

Investigating enzyme activity: the practicals

Amylase and starch, followed with iodine. Amylase digests starch into maltose. Mix amylase solution with starch solution, then every 30 seconds transfer a drop to a spotting tile containing iodine solution. Iodine turns blue-black if starch is present; when the sample stays yellow-brown, all the starch has been digested. The faster the colour changes, the faster the reaction.

Catalase and hydrogen peroxide. Catalase is an enzyme found in living tissues such as potato or liver. When hydrogen peroxide is added, catalase breaks it down into water and oxygen, and the oxygen appears as bubbles. The volume of oxygen collected per minute, or the height of the foam, measures the rate of reaction.

The effect of pH, using buffer solutions. Set up several tubes with the same volume and concentration of enzyme and substrate, and add a different buffer solution to each so that they have a range of pH values. Compare the rate in each tube. A buffer is used rather than a plain acid or alkali so that the pH stays constant throughout the experiment, instead of drifting as the reaction proceeds.

Controlling the variables. Change only the variable you are investigating. Keep the temperature, the volume and concentration of the enzyme and substrate, and the time of the experiment the same in every tube. Use a water bath to hold the temperature constant. Repeat each reading and calculate a mean, so that the results are reliable rather than the product of one odd run.