How enzymes work: the active site, specificity and denaturing

Why does fresh pineapple stop a jelly from setting, when tinned pineapple lets it set perfectly? Same fruit, same jelly, same fridge. The fresh one gives you a bowl of sweet soup. The tempting answer is that fresh fruit is juicier and waters the jelly down. But tinned pineapple sits in juice too. What the tinned fruit has been through is heat. It was heated when it was canned. What that heat changed is the second half of this story; the first half is a molecule called an enzyme.

An enzyme is a biological catalyst. A catalyst speeds up a reaction and is not used up by it. That means one enzyme molecule can do the same job again and again. Enzymes are proteins, built at the ribosomes, where protein synthesis happens. And a protein's job comes from its shape. Every enzyme has a dent in its surface with a particular shape. That dent is the active site, and it is the part that does the work. The molecule an enzyme works on is its substrate. The enzyme has the dent; the substrate is the smaller molecule that goes into it. Take amylase, an enzyme that breaks starch down into sugar. Amylase is the enzyme, with the active site. Starch is its substrate. For a substrate to go in, its shape must be complementary to the active site. Complementary means the two shapes fit together, bump into dip. Three molecules sit beside one enzyme. The first is round, the second is a long rod, and the third is a block with a step cut into one side. Which of the three can go into this active site, and why that one? The block with the step. Its shape is complementary to the dent, so it fits. Then two things happen, one after the other. First, the substrate fits into the active site. Second, it binds there, held in place. Fits, then binds: they are two separate steps, and an answer needs both. While the two are joined, the pair has a name: an enzyme-substrate complex. In the complex, the substrate is broken apart, or joined to another molecule. Then the products leave, and the enzyme is free for the next one. Now a why: the round molecule bumps into this enzyme. Why is it never broken down? It is not complementary to the active site, so it cannot fit. If it never fits, it never binds, and no complex forms. One examiner's report, on a Higher paper, records two slips about enzyme and substrate. Several students thought that starch was an enzyme or that amylase fitted into the enzyme. And many stated that the substrate fits into the active site, but did not go on to say that binding occurred. The fix is two habits. Keep enzyme and substrate as two words for two different things, and always write both steps: it fits, then it binds.

One enzyme works on only one kind of substrate. That is what specific means. The reason is shape. Only a substrate that is complementary to the active site can fit and bind, so every other molecule is left alone. A simplified model pictures this as a lock and a key. Which one plays the enzyme in that model, the lock or the key, and why? The lock, because the lock has the shaped hole, and only the enzyme has an active site. The substrate is the key. It is a model, not a photograph. Real active sites are a little more flexible than metal, but the rule that shape decides the fit holds. Now two enzymes and two substrates, each drawn with its shape: catalase and amylase, hydrogen peroxide and starch. Catalase with hydrogen peroxide: the shapes are complementary, so it fits, binds, and is broken down into water and oxygen. Swap the partners round and test each pair the same way. Catalase with starch: is there a reaction, and why or why not? No reaction. Starch is not complementary to the active site of catalase, so it cannot fit. The last two pairs follow the same rule. Amylase with starch fits, so starch is broken down. Amylase with hydrogen peroxide does not fit, so nothing happens. Now step back: if each enzyme fits one substrate, how many enzymes does a cell need? One for each reaction it runs, and it runs thousands of different reactions, so it needs thousands of different enzymes. Here is one examiner's report, from a Higher paper, on a question about hydrogen peroxide. Examiners were looking for ideas covering the specific shape of the active site and the fact that only hydrogen peroxide molecules can fit the active site. Some candidates did not mention the active site and only referred to the shape of the enzyme. Others confused the lock and key process and thought that the enzyme entered the active site of the hydrogen peroxide molecule. Two answers went wrong there. One talked about the whole enzyme, not its active site. The other ran the model backwards, putting the dent in the substrate. The fix: name the active site every time, and keep the dent in the enzyme. The substrate goes into the active site, never the other way round.

If its active site is bent out of shape, an enzyme stops working. An enzyme like that is denatured. Denatured means three linked steps, in this order. The active site changes shape. Then the substrate no longer fits. So the reaction stops. Two things denature an enzyme: a temperature that is too high, or a pH too far from the pH it works best at. Now a prediction: an enzyme is kept in a fridge overnight. Is it denatured? No. Cold slows an enzyme down, but its active site keeps its shape, and warming it brings it back. The video on what changes the rate of an enzyme-controlled reaction draws the whole curve. Denaturing changes a shape; it does not make the enzyme disappear. The enzyme is still there, still a protein, with the wrong shape. Here is a real case. A dairy uses the enzyme lactase to break down lactose, the sugar in milk, in a tank kept at pH six point five. A valve fails, and a batch of milk arrives at pH three point five. The lactase in it does nothing at all. Explain why in three linked steps, starting from the pH and ending at the substrate. The low pH makes the active site of lactase change shape. Lactose then no longer fits, so no enzyme-substrate complexes form. The lactase is denatured. Now the pH is put back to six point five. Does that same lactase work again? No. Putting the pH right does not reshape the active site, so the lactase stays denatured. That is the difference from cold, which only slows an enzyme down. Keep one kind of word out of your answers: any word that treats an enzyme as alive. An enzyme is a molecule, and it was never alive. One examiner's report, on a Foundation paper, sets the word down plainly. A misconception has been identified as some students did not understand the term denatured, which is when the active site changes shape and the substrate no longer fits. The way to show you understand it is to write the steps as well as the word: shape changes, substrate no longer fits, reaction stops. Three students explain why a boiled potato extract no longer breaks down hydrogen peroxide. The first, answer A, says the enzyme was killed by the heat. The second, answer B, says the enzyme melted, so it could not work. The third, answer C, says the high temperature changed the shape of the active site, so hydrogen peroxide no longer fits. Which of the three gets the mark, and what is missing from the other two? Only C. Answer A uses a word that tells the examiner nothing about the active site. B fails the same way: melted is another wrong word, and it names no active site and no substrate. Now fix answer A, changing as few words as you can. The heat denatured the enzyme: its active site changed shape, so hydrogen peroxide no longer fits. There is encouragement in another report, from a Higher paper, about this same chain. Most candidates linked pH five being too acidic to causing the enzyme to denature and the active site to change shape. Link the cause to the change of shape like that, and you are writing what most of those students wrote.

Fresh pineapple holds an enzyme named bromelain. Its substrate is protein, and it breaks protein down. A jelly sets because of gelatin, and gelatin is a protein. Bromelain breaks it down, so the fresh-fruit jelly never sets. Now picture the same jelly, made this time with fruit from the tin. Why does that jelly set? Answer with the word denatured. Canning heated the fruit and denatured the bromelain. Its active site changed shape, so gelatin no longer fits, and the jelly sets.

The pineapple jelly is explained. Three quick checks before you go, and the first uses an enzyme this video never described. Lipase breaks down fats. Will it break down starch, and why? No. Starch is not complementary to the active site of lipase, so it cannot fit or bind. Now a chain: an enzyme is boiled. Why does it stop working, in three steps? Its active site changes shape, so the substrate no longer fits, so the reaction stops. The enzyme is denatured. One more: fresh pineapple is chilled overnight before it goes into a jelly. Will the jelly set? No. Cold only slowed the bromelain. Its active site kept its shape, so it still breaks the gelatin down.

If you've got this down, give it a thumbs-up and cross it off your list. If active sites still feel slippery, save it for now; the rest of this chapter keeps using these words, so they come back round.

Next in the chapter: What changes the rate of an enzyme-controlled reaction. Hopefully that's next in your feed, but if not, search for ScholaFly B I oh three, oh two.

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