AP BIOLOGY · UNIT 3 · CELLULAR ENERGETICS

How enzymes run a cell

In a test tube, some of the reactions inside you would take years. In a cell they are over in a thousandth of a second. This is how enzymes do it, what slows them down, and where the energy for life comes from.

AP Biology 3.1 to 3.33 lessons8 chaptersdry lab

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By the end you can tap a goal to go to its chapter

Explain how the shape and charge of an active site make an enzyme specific to its substrate 3.1.A chapter 1 →
Explain how an enzyme speeds up a reaction by lowering its activation energy, and why it leaves ΔG unchanged 3.1.A chapter 1 →
Explain how heat and extreme pH change an enzyme's structure, and when that change can be undone 3.2.A chapter 4 →
Predict how temperature, pH, substrate and product concentrations and inhibitors change the rate of a reaction 3.2.B chapter 3 →
Describe why a living cell needs a constant input of energy, and how ATP couples an uphill reaction to a downhill one 3.3.A chapter 7 →
Explain why energy pathways run in small steps, and what the same pathways in every domain of life tell us 3.3.B chapter 8 →

01Learn

Eight chapters. Read one, play its figure through or drag its slider, and make each prediction before you open the answer.

1The active site fits one substrate
2Enzymes lower the activation energy
3More substrate, more collisions, up to a limit
4Heat makes it faster, then breaks it
5pH changes the charges in the site
6Inhibitors block the site, or bend it
7Staying alive takes a constant supply of energy
8Pathways go in small steps, shared by all life

3.1.A Enzyme structure

The active site fits one substrate

An enzyme is a biological catalyst: a protein that makes one reaction go faster and is not used up by it. One molecule of catalase, the enzyme in this guide's dry lab, breaks down millions of hydrogen peroxide molecules every second, for as long as its shape lasts.

That shape is everything. An enzyme is a chain of hundreds of amino acids folded into a precise 3D shape, held there by hydrogen bonds and ionic bonds between R groups. A small pocket in the fold, lined by just a few of those amino acids, is the active site. The molecule the enzyme works on is its substrate.

A substrate binds only if two things match: its shape fits the site, and its charges are opposite to the charged R groups lining it. That double check is why an enzyme is specific. As the substrate settles in, the site shifts to grip it more tightly, which strains the bond that is about to break: induced fit.

Enzyme names usually end in -ase and name the substrate: lactase splits lactose, lipase splits lipids.
Shape and charge both have to match, or there is no binding and no reaction.
The enzyme lets go of the products unchanged and binds the next substrate.

Watch out The substrate and the active site are not "the same shape". They are complementary: the site is the mold and the substrate fills it, as a hand fills a glove. And the site is not a rigid lock. It moves as the substrate binds.

Example Lactase splits lactose, which is glucose joined to galactose. Sucrose is glucose joined to fructose: exactly the same atoms as lactose, arranged differently. Lactase leaves it alone: the site grips the galactose half, and sucrose has none.

A molecule has exactly the substrate's shape, but its two charges are the other way round. What happens at the active site?

It binds. Shape fits and the charges attract, so the substrate settles in and the loops close round it: induced fit.

Offer the enzyme each molecule, then tap a part to read about it.

3.1.A Enzyme catalysis

Enzymes lower the activation energy

Breaking a bond always takes energy in; forming a bond releases energy. So even a reaction that releases energy overall must first be pushed over a hump, while old bonds strain and start to break. That push is the activation energy. At body temperature very few collisions carry enough of it, so most reactions in a cell would crawl.

An enzyme gives the reaction a lower route. Its active site holds the substrate in exactly the right position, induced fit bends the bond that has to break, and charged R groups pull electrons the right way. Less energy is needed, so far more molecules get over the top each second.

What the enzyme cannot change is where the reaction starts and where it ends. The free energy of the reactants and of the products, and so the difference between them, ΔG, are the same with or without it. It changes the route, not the destination, and it speeds the reverse reaction just as much.

The enzyme lowers the activation energy. It supplies no energy of its own.
ΔG does not change, so an enzyme cannot make an uphill reaction go by itself.

Watch out "Breaking bonds releases energy" is wrong, and it costs marks every year. Breaking a bond always takes energy in. Energy is released when new, more stable bonds form, and the enzyme only lowers the cost of getting started.

Example One step in making the nucleotides of DNA and RNA has, with no enzyme, a half-life of about 78 million years. Its enzyme, orotidine phosphate decarboxylase, gets each molecule through in about a fortieth of a second: a speed-up of around 10¹⁷.

An enzyme is added to a reaction with ΔG = −20 kJ/mol. What is ΔG now?

The reactant must pass over a hump before it can become products. Here the products end up lower: this reaction releases energy.
At the top of the hump the bond is stretched and about to break. Getting there takes the activation energy, and at 37 °C few collisions have that much.
With the enzyme the route is lower: the active site holds the substrate in place and strains its bond, so less energy is needed.
The start and the finish have not moved, so ΔG is the same with or without the enzyme. The enzyme changes the route, never the destination.
A lower peak means many more molecules have enough energy to cross it each second, so the reaction runs far faster.

3.2.B Substrate and enzyme concentration

More substrate, more collisions, up to a limit

Enzyme and substrate molecules jostle about at random. A reaction can only happen when a substrate meets a free active site the right way round, so the rate depends on how often that happens.

Add more substrate and those meetings happen more often, so the rate climbs. But each enzyme molecule can only work so fast. Once there is so much substrate that every active site is refilled the moment it empties, the enzyme is saturated and the rate levels off at its maximum. Past that point, only more enzyme raises it.

Products matter too. As a reaction runs, substrate is used up and product builds up, so the rate falls: the relative concentrations of substrate and product set how fast it goes. Many reactions are reversible, and the same enzyme speeds whichever direction the concentrations favor.

Low substrate: the rate is limited by how often substrate meets a free site.
High substrate: every site is busy, and the rate is limited by the amount of enzyme.

Watch out The plateau is not the enzyme being used up or denatured. Every enzyme molecule is still there and still working, flat out. Add more enzyme and the rate climbs again.

Example Carbonic anhydrase in red blood cells turns CO₂ and water into hydrogen carbonate (HCO₃⁻) as they pass through your muscles, where CO₂ is high. In your lungs, where CO₂ is low, the same enzyme turns it back into CO₂ to breathe out.

Remember it The five things that change an enzyme's rate spell SPITE

SpicySubstratehow much there is
PepperspHcharges in the site
IrritateInhibitorsblock or bend the site
TiredTemperaturecollisions, then unfolding
EyesEnzymehow much there is

A reaction has reached its plateau: more substrate no longer speeds it up. Which change would raise the rate?

Substrate6

Drag the substrate up a step at a time and watch the active sites fill. Each concentration you try adds a point to the graph.

3.2.A 3.2.B Temperature

Heat makes it faster, then breaks it

Warm a solution and its molecules move faster. Enzyme and substrate collide more often, and more of those collisions carry enough energy to react, so the rate rises. For many enzymes it roughly doubles with every 10 °C.

But an enzyme's shape is held by weak bonds, mostly hydrogen bonds. Past a point, its atoms vibrate so hard that these bonds break: the chain loosens, the active site loses its shape, the substrate no longer fits, and the rate falls steeply. An enzyme whose structure has been disrupted like this is denatured. The temperature where the two effects balance and the rate peaks is the optimum.

Denaturing breaks the bonds between R groups, not the peptide bonds, so the amino acid sequence survives. Sometimes, when conditions return to normal, the chain folds back and the enzyme works again: ribonuclease, unfolded by chemicals, refolds fully active once they are washed away. Chains unfolded by heat usually tangle and stick together instead, and the loss is permanent.

Rising side: more collisions, and more of them with enough energy.
Falling side: denaturation, faster the hotter it gets.
Each enzyme's optimum suits the place it works.

Watch out Cold does not denature an enzyme. At 5 °C it is slow because it and its substrate move slowly, but its shape is intact, and it works normally once warmed. Write "denatured" for the hot side only (or an extreme pH).

Example Most human enzymes peak near 37 to 40 °C. The DNA polymerase of Thermus aquaticus, a bacterium from hot springs, works best at about 75 °C, which is why PCR uses it. Antarctic icefish have enzymes that work in water at about 0 °C.

Salivary amylase is held at 70 °C for ten minutes, then cooled back to 37 °C. What is its rate now?

Temperature37

Drag the temperature up past 45 °C and watch active sites change shape for good; add substrate until every site is busy; then try each inhibitor.

3.2.A 3.2.B pH

pH changes the charges in the site

pH measures how many hydrogen ions (H⁺) a solution holds: pH = −log[H⁺]. The scale is logarithmic, so each step down means ten times more H⁺, and pH 4 holds a thousand times the H⁺ of pH 7. You need the idea, not the calculation.

Many R groups in an active site can gain or lose H⁺. A carboxyl group (–COO⁻) picks one up in acid and loses its negative charge; an amino group (–NH₃⁺) gives one up in alkali and loses its positive charge. Change those charges and the substrate is no longer held, so the rate drops. Push the pH further and the ionic and hydrogen bonds that hold the fold break too: the enzyme denatures.

So every enzyme has an optimum pH, and it suits the place the enzyme works: pepsin in the acid of the stomach, trypsin in the small intestine, where alkaline juice from the pancreas has neutralized that acid.

A little off the optimum: charges in the site change, and the effect reverses when the pH returns.
Far off it: the bonds holding the fold break, and the enzyme denatures.

Watch out "Enzymes work best at pH 7" is not true of all of them: pepsin barely works at pH 7. And one pH unit is not a small change. It is ten times the H⁺.

Example Pepsin starts digesting protein in the stomach at about pH 2. When food moves on into the small intestine, pancreatic juice at about pH 8 neutralizes the acid. Pepsin stops working there, and trypsin, which arrives in that juice and works best near pH 8, takes over.

Amylase is put in a buffer at pH 5 for a minute, then moved back to pH 7. What is its rate now?

pH7

3.2.B Inhibitors

Inhibitors block the site, or bend it

An inhibitor is a molecule that binds to an enzyme and slows it down. There are two ways to do it, and a graph of rate against substrate concentration tells them apart.

A competitive inhibitor looks enough like the substrate to fit the active site, and while it sits there the substrate cannot bind. Most come and go, so inhibitor and substrate take turns at the sites, and the more substrate there is, the more often the substrate wins. Add enough and the rate climbs back to nearly its normal maximum.

A noncompetitive inhibitor binds somewhere else, at an allosteric site. It changes the shape of the enzyme, active site included. The substrate is not competing for that spot, so no amount of substrate wins those enzymes back: the maximum rate stays lower.

Most inhibitors bind reversibly and let go again. Some bond to the enzyme for good and take it out of action permanently, whichever site they use.

Competitive: same maximum rate, but more substrate is needed to reach it.
Noncompetitive: a lower maximum rate, whatever the substrate concentration.

Two reactions are slowed by inhibitors. You add a large excess of substrate to both. One gets back almost its full rate; the other does not. What does the one that recovered contain?

Examples Zanamivir (Relenza), a flu drug, is shaped like sialic acid. New flu virus particles stay stuck to the cell they leave until the virus's enzyme neuraminidase cuts away the sialic acid they cling to. Zanamivir fills that enzyme's active site, so they stay stuck: a competitive inhibitor. Penicillin binds the active site of the enzyme bacteria use to cross-link their cell walls, and bonds to it for good. Isoleucine shuts down its own production by binding an allosteric site (chapter 8).

Watch out Cyanide is often given as an allosteric inhibitor. It is not one: it binds the iron and copper center of cytochrome c oxidase, the very place where oxygen should bind at the end of the electron transport chain. And a competitive inhibitor does not lower the maximum rate. It raises the substrate concentration needed to reach it.

Remember it Two kinds of inhibitor, and where the second binds

CopycatCompetitivetakes the active site; more substrate wins
NopeNoncompetitivebends the site; max rate drops
AnotherAllosteric sitea site that is not the active site

Substratelow

3.3.A Cellular energy

Staying alive takes a constant supply of energy

A cell is a highly ordered place: proteins folded just so, membranes whole, more potassium inside than out. The second law of thermodynamics says that every energy transfer turns some energy into heat, and the disorder of the universe as a whole always increases. Order never keeps itself. A cell holds on to it only by taking in energy all the time, from food or from light, faster than it loses energy. Cut off that flow, or lose the order, and the cell dies.

Life does not break the second law. A cell's order is paid for by the heat and disorder it releases into its surroundings.

Reactions come in two kinds. An exergonic reaction releases free energy (ΔG is negative), like the breakdown of glucose. An endergonic reaction needs free energy put in (ΔG is positive), like building a protein. A cell runs an endergonic reaction by coupling it to an exergonic one, and the usual partner is the hydrolysis of ATP, the cell's energy currency.

Energy in must exceed energy lost, to keep order and to do work.
Coupled to ATP hydrolysis, an uphill step becomes part of a downhill pair.

Watch out Energy is not stored in ATP's last phosphate bond like a spring, released when the bond snaps. Breaking any bond takes energy in. ATP hydrolysis releases energy because the products, ADP and phosphate, are more stable than ATP and water were: they hold less free energy.

Example Your body holds only about 250 grams of ATP at any moment, yet makes and spends roughly its own weight in ATP every day. Each molecule is split to ADP and rebuilt, over and over, with energy from respiration.

Remember it Which way the energy goes: read the first two letters. EXergonic, energy EXits; ENdergonic, energy ENters.

ExitsExergonicenergy leaves; ΔG negative
EntersEndergonicenergy goes in; ΔG positive

Reaction X has ΔG = +20 kJ/mol. Which of these would make it run in a cell?

Order kept. Energy coming in rebuilds what wears out: molecules stay built, proteins folded, ions pumped inside. Heat flows out.

Making glutamine from glutamate and ammonia is uphill: ΔG = +14 kJ/mol. On its own it hardly happens.
Splitting ATP into ADP and phosphate is downhill: ΔG = −31 kJ/mol. The products are more stable than ATP was.
The enzyme glutamine synthetase couples them: ATP hands its end phosphate to glutamate. Glutamyl phosphate is unstable, ready to react.
Ammonia takes the phosphate's place, making glutamine. Overall +14 − 31 = −17 kJ/mol: downhill, so it runs. Respiration then rebuilds the ATP.

Standard values. In a living cell, splitting ATP releases even more, about −50 kJ/mol.

3.3.A 3.3.B Energy pathways

Pathways go in small steps, shared by all life

Cells rarely do anything in one reaction. They use metabolic pathways: chains of reactions, each catalyzed by its own enzyme, where the product of one step is the substrate of the next. Set glucose alight and its energy comes out all at once, as heat and light, and none of it is kept. Broken down in a cell, it goes through dozens of steps, each releasing a little energy that the cell can capture as ATP and control.

Control often acts on the first step. When the end product builds up, it binds an allosteric site on the pathway's first enzyme and switches it off: feedback inhibition. When the product is used up it lets go, and the pathway runs again. No energy or raw material is wasted making what the cell already has.

The same core pathways turn up everywhere. Bacteria, archaea and eukaryotes all use ATP as their energy currency, and versions of glycolysis run in all three, with several of the same enzymes. Pathways shared and conserved like this are best explained by inheritance from a common ancestor of all living things.

The product of one step is the substrate of the next.
The end product inhibits the first enzyme: negative feedback.
Conserved pathways in every domain are evidence of common ancestry.

Watch out Feedback inhibition does not destroy the first enzyme. The end product binds reversibly: as its level falls it lets go, and the enzyme works again. That is what makes it a switch.

Example Your liver breaks alcohol down in steps: ethanol to acetaldehyde (alcohol dehydrogenase), acetaldehyde to acetate (aldehyde dehydrogenase), then into the Krebs cycle. Some people inherit a slow version of the second enzyme. Toxic acetaldehyde builds up, and their face flushes after a drink.

The end product of a pathway builds up in a cell. What happens to the first enzyme of that pathway?

A pathway: each product is the next enzyme's substrate, so threonine is turned into isoleucine step by step (three steps here; the real pathway has five).
The cell makes more isoleucine than it is using, and it builds up.
Isoleucine binds an allosteric site on enzyme 1 and bends its active site. The first step stops, so the whole pathway stops.
As the cell uses isoleucine to build proteins, the level falls, isoleucine lets go, and the pathway runs again: negative feedback.

Bacteria, archaea and eukaryotes all run on ATP, and all break glucose down by versions of glycolysis, several of whose enzymes have closely similar sequences in all three.
All three groups trace back to one common ancestor. Archaea and eukaryotes share a more recent one as well.
The simplest explanation: the ancestor already had ATP and glycolysis, and every line of descent kept them. Shared, conserved pathways are evidence of common ancestry.

02Dry lab: Catalase and hydrogen peroxide

Catalase, from a cell extract, breaks hydrogen peroxide down to water and oxygen. Collect the oxygen in a gas syringe, find the initial rate, and see what changes it.

Your questionHow does one factor change the rate at which catalase breaks down hydrogen peroxide? Start with temperature: where is the optimum, and what happens beyond it?

What you can changeOne independent variable at a time: temperature, pH, hydrogen peroxide concentration, extract concentration, or an inhibitor, competitive or noncompetitive. Everything else stays fixed unless you change it on purpose.

What to recordThe volume of oxygen every 10 seconds, then the initial rate in cm³/min: the gradient at the very start. Three trials at each value, a mean, and a graph of the means.

Task 1

Find the optimum temperature

Choose Temperature. Run 0, 10, 20 and so on up to 80 °C, three trials at each, and press Add to table after every run. Plot the means, then go back and fill in every 5 °C around the peak.

Record: the optimum to the nearest 5 °C, and how a run at 65 °C or above behaves after its first 30 seconds.

Task 2

Saturate the enzyme

Choose Substrate and run hydrogen peroxide from 0.25% to 10%. Find where doubling the concentration stops doubling the rate. Then set Extract concentration to 50% under Kept the same and run the series again.

Record: where each curve levels off, and how halving the enzyme changes the plateau.

Task 3

Outcompete an inhibitor

Choose Substrate and run a series with no inhibitor. Under Kept the same, set Inhibitor to Competitive at 1 mM and run the series again; then Noncompetitive at 1 mM. Compare all three at 0.5% and at 10% peroxide.

Record: each series' rate at 0.5% and at 10%, as a percentage of the no-inhibitor rate.

Task 4

Run the controls

At 25 °C and pH 7, run Boiled extract and Water, no extract beside a normal run.

Record: the oxygen each gives in 3 minutes, and what each control rules out.

At 65 °C the oxygen curve rises for the first minute, then flattens, far below the volume the peroxide could give. Why?

In Task 3, the competitive inhibitor costs a lot of rate at 0.5% peroxide but little at 10%. The noncompetitive one halves the rate at both. Why the difference?

Why does the lab ask for the initial rate, not the total oxygen after 3 minutes?

03Check

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05Teacher resources

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Lesson	Covers	Focus	Chapters	Lesson plan	Slides
1What an enzyme does	3.1.A60 min	Specificity from shape and charge, and lowering the activation energy, with a clay model and a gas demo	1, 2	Lesson planPDF 676 KB	SlidesPPTX 701 KB
2What changes the rate	3.2.A, 3.2.B60 min	Temperature, pH and substrate concentration, tested in a circus of practicals and explained with the figures	3, 4, 5	Lesson planPDF 749 KB	SlidesPPTX 4.5 MB
3Inhibitors, pathways and energy	3.2.B, 3.3.A, 3.3.B60 min	Competitive and noncompetitive inhibition, feedback in pathways, and coupling with ATP, ending on a timed free response	6, 7, 8, dry lab	Lesson planPDF 743 KB	SlidesPPTX 1.5 MB

PDFEvery lesson planAll three plans in one PDF, with this overview at the front. 1.2 MB KEYNote template, filled inThe class’s note template with every gap filled. 1.0 MB

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Lesson: Enzymes
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Quiz: Environmental Impacts on Enzyme Function
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