Enzymes at Work

Stir pieces of fresh pineapple into gelatin and chill it, and the gelatin can stay runny. Use canned pineapple instead, and it sets. Gelatin is a protein. When it cools, its long chains tangle into a net that traps water, and the dessert turns firm. A University of Georgia Extension food lab uses this contrast to ask a question: what is in fresh pineapple, and what did canning change?

Explore: speed up and slow down an enzyme

In the enzyme rate explorer you control one enzyme in a test tube. Sliders set the temperature, the pH and how much substrate is in the tube, and a graph shows how fast the reaction runs. The curves are a sketch of the usual pattern, not measured data for any one real enzyme.

Try this.

1. Set the pH in the middle and the substrate high. Predict what the rate will do as you slide the temperature from cold to hot. Then test it.
2. Find the temperature with the highest rate. Predict whether the peak stays as tall when you move the pH far to one side. Then test it.
3. Keep temperature and pH at their best values. Predict whether doubling the substrate doubles the rate every time. Then double it several times.
4. Push the temperature very high, then bring it back down. Predict whether the rate comes back, then see what the model shows.

Explain: how an enzyme speeds up a reaction

Drop a piece of raw potato into hydrogen peroxide, the kind sold in brown bottles at a drugstore, and it fizzes. Hydrogen peroxide breaks down by itself, but slowly. Something in the potato makes it break down fast, and the bubbles are oxygen gas.

hydrogen peroxide → water + oxygen

2 H₂O₂ → 2 H₂O + O₂ (sped up by catalase)

The something is catalase, found in potato cells, liver cells and other animal cells. Your cells run a huge number of chemical reactions like this one. Metabolism is all the chemical reactions in a living thing. Many of them would be far too slow at body temperature without help.

The help comes from enzymes: molecules, almost all of them proteins, that speed up specific reactions in living things. An enzyme is a catalyst, a substance that speeds up a reaction without being used up.

• The substrate is the molecule an enzyme acts on. For catalase it is hydrogen peroxide.
• The substrate fits into the active site, a small pocket on the enzyme where the reaction happens.
• The enzyme changes the substrate into products, the molecules a reaction makes. For catalase they are water and oxygen.
• The products leave. The enzyme is unchanged and can take in another substrate molecule.

The active site’s shape and electric charges fit only certain molecules. So each enzyme usually speeds up one reaction, or a few similar ones. Bromelain in pineapple cuts protein chains. Catalase does not touch protein; it splits hydrogen peroxide.

A common model is a lock and key: the substrate is a key that fits one lock, the active site. The model breaks in two places. A real enzyme is not rigid. When the substrate binds, the enzyme often shifts its shape a little to grip it more tightly, which biologists call induced fit. And charge matters as well as shape.

Why does fitting into a pocket make a reaction faster? Picture a ball that must be pushed over a hill before it can roll down the other side. Activation energy is the energy needed to get a reaction started, like that hill. An enzyme does not add energy. It holds the substrate in a position where bonds break or form more easily, which lowers the hill. With a lower hill, many more substrate molecules react each second at body temperature. The analogy has a limit: the hill is a graph of energy, not a real place.

A few catalysts in cells are made of RNA instead of protein. You can meet them in Biology II.

Explain: why temperature, pH and substrate matter

An enzyme works only while its active site keeps its shape. Three conditions change how fast it works.

Temperature. Warmer molecules move faster and bump into each other more often, so up to a point a warmer reaction runs faster. But a protein enzyme is a folded chain held in shape by many weak attractions. Too much heat shakes those attractions apart. To denature is for a protein to lose the folded shape it needs to work. A denatured enzyme is not dead. It is a molecule whose active site no longer fits. Heat damage is often permanent, the way a cooked egg white stays white.

The optimum is the condition at which an enzyme works fastest. Most human enzymes have an optimum near 37 °C, your body temperature. Enzymes from heat-loving microbes can have much higher ones.

This answers half the pineapple puzzle. Fresh pineapple contains bromelain, which cuts gelatin’s protein chains so they cannot tangle into a net. Canning heats the fruit enough to denature bromelain, so canned pineapple leaves the gelatin alone.

pH. pH is a scale from 0 to 14 that measures how acidic or basic a solution is; 7 is neutral. Changing the pH changes the electric charges on the active site and the substrate, so they may no longer attract each other. Very acidic or very basic conditions can also unfold the enzyme for good.

Substrate concentration. With a fixed amount of enzyme, adding a little substrate speeds the reaction, because substrate meets active sites more often. Keep adding, and the rate levels off. Saturation is the point at which nearly every active site is busy, so more substrate barely raises the rate. Picture a store with four checkout lanes: more shoppers mean more sales, until every cashier is busy. The comparison breaks because cashiers get tired and enzymes do not. Only more enzyme raises the top rate.

Elaborate: an enzyme tested at five temperatures

Scientists made an enzyme called Sfβgly in bacteria, using the gene of the fall armyworm caterpillar, purified it, and measured how active it was at five temperatures. They followed each reaction for a long time and worked out the activity at the 20 minute mark and again at the 60 minute mark. Every other condition stayed the same.

Source: Almeida and Marana, PLOS ONE, 2019, supplementary S1 Table. One lab sample of purified enzyme, with the same substrate concentration (6 mM, millimolar) at pH 6. In each series the highest value is 100%. This is not catalase, and it is a test-tube measurement, not a field measurement.

1. In the 20-minute tests, which temperature gave the highest activity? What happened between 42 and 46 °C?
2. Compare the two rows for 42 °C. By how many percentage points did activity fall from the 20-minute test to the 60-minute test?
3. Each value is the mean of three tests. What does ”± 10” at 42 °C tell you, and why is a mean of three more trustworthy than one test?

Design problem

Plan a fair test of how temperature changes the rate of catalase from potato. Name your independent and dependent variables, what you keep constant, your control and how many trials you run. Say how you will stay safe.

Evaluate: check your thinking

Fresh pineapple contains bromelain, an enzyme that cuts protein, so gelatin’s chains are cut before they can tangle into a firm net. Canning heats the fruit and denatures bromelain, and a denatured enzyme no longer fits its substrate, so the gelatin sets.

Science practices you used: making predictions and testing them with a model, planning a fair test with a control and repeated trials, reading a data table of means with their spread, and naming the limits of a data set and a model.