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B. Biochemistry · Biochemistry

Controlling Enzymes: Inhibitors, Allosteric Sites and Feedback

B3.4 B2.1 A1.8 A1.12About 70 minutes
Learning goals
  • I can distinguish competitive, non-competitive and irreversible inhibition and predict their effects on reaction rate.
  • I can explain allosteric regulation, including allosteric activators and inhibitors.
  • I can describe feedback inhibition and explain why it is an efficient way to control a metabolic pathway.
  • I can use rate data to classify an inhibitor, and connect inhibition to medicines and poisons.

Words to know

inhibitor
A molecule that reduces an enzyme's activity.
competitive inhibition
Inhibition by a molecule that resembles the substrate and binds the active site, competing with the substrate. It can be overcome by raising the substrate concentration.
non-competitive inhibition
Inhibition by a molecule that binds somewhere other than the active site and changes the enzyme's shape. Adding more substrate does not overcome it.
allosteric site
A binding site on an enzyme, separate from the active site, where a regulatory molecule binds and changes the enzyme's shape and activity.
allosteric activator / inhibitor
A molecule that binds an allosteric site and stabilizes the active form (activator) or the inactive form (inhibitor) of an enzyme.
irreversible inhibitor
An inhibitor that binds an enzyme permanently, usually by a covalent bond.
feedback inhibition
Control of a metabolic pathway in which the end product inhibits an enzyme early in the pathway, usually the first.
metabolic pathway
A series of reactions, each catalyzed by its own enzyme, in which the product of one reaction is the substrate of the next.

Brakes and accelerators

A car with an engine but no brakes is dangerous. A cell with enzymes but no way to control them would be just as bad: it would waste energy making things it doesn't need, or run out of something essential. Cells switch enzymes on and off in seconds, and many of the medicines in your family's cupboard work by switching off one particular enzyme.

This lesson is about how enzyme activity is regulated and inhibited.

Remember from Grade 11

In SBI3U you learned that each enzyme has an optimum temperature and pH, that high temperature denatures it, and that some molecules called inhibitors block or change the active site. Digestive proteases are made as inactive precursors (pepsinogen, trypsinogen), another way of controlling enzymes.

Competitive inhibition

A competitive inhibitor has a shape similar to the substrate. It binds to the active site (usually by weak bonds, so reversibly) and blocks the real substrate. Inhibitor and substrate compete for the same site, so:

  • at low substrate concentration the inhibitor wins often and the rate drops a lot;
  • at high substrate concentration the substrate out-competes the inhibitor, and the rate approaches the same Vmax as without the inhibitor (it just takes more substrate to get there: the apparent Km increases).

Examples: Statins, prescribed to lower blood cholesterol, compete with the natural substrate of a liver enzyme (HMG-CoA reductase) in the cholesterol-making pathway. Sulfa drugs (sulfonamide antibiotics) look like a molecule bacteria need to make the vitamin folic acid, and block that bacterial enzyme. In methanol poisoning, doctors may give ethanol or the drug fomepizole, which compete with methanol for the enzyme alcohol dehydrogenase, so less methanol is turned into toxic products while the body clears it.

Non-competitive inhibition

A non-competitive inhibitor binds to a site other than the active site (an allosteric site). It changes the enzyme's shape so the active site works poorly or not at all. Because it doesn't compete for the active site, adding more substrate doesn't help: Vmax is lowered.

Example: Heavy-metal ions such as mercury (Hg²⁺) and lead (Pb²⁺) bind to sulfhydryl (–SH) groups on many enzymes, changing their shape. This is one reason these metals are toxic.

Irreversible inhibition

Some inhibitors bind covalently to the enzyme, so it stays inhibited permanently; the cell must make new enzyme.

  • Aspirin attaches an acetyl group to the enzyme cyclooxygenase (COX), which makes molecules involved in pain and inflammation.
  • Penicillin binds permanently to a bacterial enzyme that builds the cell wall, so bacteria can't build their walls properly.
  • Organophosphate pesticides and nerve agents bind permanently to acetylcholinesterase, an enzyme that clears a neurotransmitter from synapses. That is why these chemicals are so dangerous and are strictly regulated.
normal bindingsubstrate fitsactive sitecompetitive inhibitorsubstrateblockedinhibitor occupiesthe active sitenon-competitive inhibitorallosteric sitecan't fitinhibitor binds elsewhere;active site changes shape
Normal binding, competitive inhibition (the inhibitor, a solid triangle, sits in the active site) and non-competitive inhibition (the inhibitor, a solid circle, binds an allosteric site and the active site changes shape). Simplified shapes.
0481216200255075100substrate concentration (mmol/L)rate (µmol/min)no inhibitorcompetitivenon-competitiveVmax (no inhibitor)
Rate vs substrate concentration with no inhibitor, a competitive inhibitor and a non-competitive inhibitor (same amount of enzyme). A competitive inhibitor can be overcome by high substrate; a non-competitive inhibitor lowers Vmax.
Worked example

Example: Classify the inhibitors

An enzyme was tested with no inhibitor, with inhibitor X and with inhibitor Y. Rates are in µmol/min.

  • [S] = 1 mmol/L: none 33, X 14, Y 17
  • [S] = 2 mmol/L: none 50, X 25, Y 25
  • [S] = 5 mmol/L: none 71, X 45, Y 36
  • [S] = 10 mmol/L: none 83, X 63, Y 42
  • [S] = 20 mmol/L: none 91, X 77, Y 45

Classify X and Y, and justify with the data.

  1. Look at what happens as [S] gets large.
  2. With X, the rate keeps climbing toward the uninhibited rate: 14 is 42% of 33 at low [S], but 77 is 85% of 91 at high [S]. More substrate is overcoming the inhibitor: X is competitive.
  3. With Y, the rate levels off near 45 to 50, about half of the uninhibited maximum, and stays at roughly half the uninhibited rate at every concentration (17 of 33, 25 of 50, 45 of 91). More substrate does not help: Y is non-competitive, lowering Vmax.
  4. A good next step (A1.8) would be to test an even higher [S], such as 50 mmol/L, to confirm that X approaches Vmax while Y stays near half of it.

X is a competitive inhibitor (overcome by high substrate; same Vmax). Y is a non-competitive inhibitor (Vmax lowered to about half).

Allosteric regulation

Many enzymes that control key steps are made of several subunits and have allosteric sites. These enzymes flip between an active and an inactive shape:

  • An allosteric activator binds and stabilizes the active shape.
  • An allosteric inhibitor binds and stabilizes the inactive shape.

The activators and inhibitors are often molecules that report on the cell's needs. For example, an enzyme early in glucose breakdown, phosphofructokinase, is inhibited by ATP (plenty of energy: slow down) and activated by AMP and ADP (energy running low: speed up). You'll see this enzyme again in Metabolic Processes.

Feedback inhibition

In a metabolic pathway A → B → C → D, each step has its own enzyme. If the end product D acts as an allosteric inhibitor of the first enzyme, then:

  • when D builds up, it shuts down the pathway at the start, so the cell stops wasting A and energy on intermediates it doesn't need;
  • when D is used up, it leaves the enzyme, and the pathway starts again.

A classic example: in bacteria, the amino acid isoleucine is made from threonine in five steps, and isoleucine inhibits the first enzyme of the pathway. This is negative feedback at the molecular level: the same idea you'll meet for whole-body control in Homeostasis.

ABCDE1E2E3end productstartingsubstrateD binds an allosteric site on E1 and switches it offlots of D →pathwayslows
Feedback inhibition: the end product D binds an allosteric site on the first enzyme (E1) and slows the whole pathway. When D is used up, the pathway restarts.
TypeBinds toOvercome by more substrate?Effect on VmaxExample
CompetitiveActive siteYesSame (needs more substrate)Statins; sulfa drugs
Non-competitiveAllosteric (other) siteNoLoweredHeavy metals on –SH groups
IrreversibleUsually active site, covalentlyNoLowered (enzyme lost)Aspirin on COX; penicillin
Allosteric activatorAllosteric site(increases activity)RaisedAMP on phosphofructokinase
Summary of the ways molecules change enzyme activity.
Just for fun

A competitive inhibitor is like someone sitting in your reserved seat at a Raptors game. Bring enough friends (substrate) and eventually you get the seats back. A non-competitive inhibitor is more like someone who folded your seat up from the row behind.

Hands-on activity

Paper-ase with inhibitors

Use the Paper-ase setup from How Enzymes Work (paper strips, a table, a timer and a partner).

  1. Baseline: run 15-second trials at 10 and 80 strips (repeat each twice, average).
  2. Competitive inhibitor: make 20 "decoy" strips the same size from thin cardboard (a cereal box) and mark each with an X. Mix the decoys in with 10 paper strips, and again with 80 paper strips. When you pick up a decoy, you must hold it for 2 seconds, then drop it unchanged (it occupies your "active site" without reacting). Run the trials.
  3. Non-competitive inhibitor: remove the decoys. Put an oven mitt on your tearing hand (the inhibitor binds elsewhere and changes your shape). Run the trials at 10 and 80 strips.
  4. Make a table and a bar graph of the rates. Calculate each inhibited rate as a percentage of the matching uninhibited rate.

What to look for: the decoys cut the rate a lot at 10 strips but much less at 80 strips (competition is overcome by more substrate). The oven mitt lowers the rate by a similar percentage at both 10 and 80 strips: your top speed (Vmax) has dropped. Discuss one way your model is not like a real enzyme (A1.8).

Your turn

1

How does a competitive inhibitor reduce an enzyme's activity?

2

Why can't adding more substrate overcome a non-competitive inhibitor?

3

What is an allosteric site?

4

Describe feedback inhibition in a pathway A → B → C → D.

5

Why is it efficient to inhibit the first enzyme of a pathway rather than the last?

6

Aspirin binds covalently to the enzyme COX. What type of inhibition is this, and how does the body recover enzyme activity?

More practice: warm-up

1

Name the three main types of enzyme inhibition.

2

Does an allosteric activator increase or decrease enzyme activity?

3

Which type of inhibitor resembles the substrate?

4

Which enzyme do organophosphate pesticides inhibit?

5

What is a metabolic pathway?

More practice: on your own

1

An inhibitor lowers the rate at all substrate concentrations, and the maximum rate it allows is 40% of the normal Vmax. Which type is it most likely to be?

2

Explain how high levels of ATP slow down glucose breakdown.

3

Why is giving ethanol a possible treatment for methanol poisoning?

4

Mercury poisoning damages many different enzymes, not just one. Use the idea of functional groups to explain why.

5

Sketch-describe how the graph of rate vs [S] changes with a competitive inhibitor.

Challenge

1

A bacterium makes amino acid Z through the pathway W → X → Y → Z. A mutation changes the allosteric site of the first enzyme so Z can no longer bind. Predict two consequences for the cell.

2

Penicillin kills many bacteria but is generally safe for human cells. Use your knowledge of enzymes and cell structure to explain why.

3

Using the example data (inhibitors X and Y), calculate the percentage inhibition caused by X at [S] = 1 and at [S] = 20 mmol/L. What does the change show?

Check your understanding

Quiz

1.

A competitive inhibitor:

2.

In feedback inhibition, the molecule that inhibits the first enzyme of a pathway is usually:

3.

A non-competitive inhibitor affects the rate curve by:

4.

An allosteric activator:

5.

Which is an example of irreversible inhibition?

6.

Statins lower cholesterol by competing with the substrate of a liver enzyme. Raising the concentration of that substrate would:

For parents and teachers

Big idea: This lesson completes B3.4 (enzyme mechanisms and regulation) and uses the B2.1 terms allosteric site and inhibition. It practises judging whether evidence supports a conclusion (A1.8) and reading graphs (A1.12).

How to help: Use the seat analogy from the fun box: ask your teen which kind of inhibitor each one models and why. The inhibitor version of Paper-ase needs a partner and an oven mitt.

Common difficulties:

  • Thinking competitive inhibitors lower Vmax. They don't; they make it harder to reach.
  • Thinking "allosteric" always means inhibition. Allosteric molecules can activate or inhibit.
  • In feedback inhibition, saying the end product inhibits the last enzyme. It is usually the first.
  • Safety note: the methanol example is about hospital treatment. Methanol, pesticides and heavy metals are dangerous and must never be handled at home.