Enzyme Inhibition, Cofactors, and Coenzymes

Enzyme inhibitors are molecules that reduce or stop enzyme activity, either by competing with the substrate for the active site (competitive inhibition) or by binding elsewhere and changing the enzyme’s shape (non-competitive inhibition), while cofactors and coenzymes are non-protein helpers that some enzymes need to work. In brief: in competitive inhibition, adding more substrate can overcome the inhibitor, but in non-competitive inhibition it cannot. Metal ions such as Zn²⁺ and Mg²⁺ are cofactors, and organic helpers derived from vitamins, such as NAD⁺, are coenzymes.

Inhibition is a natural way to control enzymes in the cell and the basis of many drugs and poisons. This guide explains each type of inhibition, shows how they affect the reaction rate, describes allosteric regulation, explains the roles of cofactors and coenzymes, works through a competitive inhibition example with numbers and corrects common mistakes.

What is enzyme inhibition?

An inhibitor is a substance that decreases the rate of an enzyme-catalyzed reaction. Inhibition can be reversible, when the inhibitor binds by weak bonds and can detach, or irreversible, when it binds permanently, usually by covalent bonds. Cells use reversible inhibitors to regulate metabolism, as in feedback inhibition. Irreversible inhibitors are often poisons.

What is competitive inhibition?

A competitive inhibitor has a shape similar to the substrate. It enters the active site, and blocks the substrate from binding, but it is not converted to product. The inhibitor and the substrate compete for the same active sites, and the proportion of enzyme molecules blocked at any time depends on the relative concentrations.

• Increasing the substrate concentration reduces the effect of the inhibitor, because the substrate outcompetes it. The same maximum rate (V_max) can be reached at a high enough substrate concentration, but more substrate is needed to reach half of V_max, so K_m appears larger.
• Example: malonate is a competitive inhibitor of succinate dehydrogenase, an enzyme of the Krebs cycle, because its structure is similar to that of succinate, the substrate.
• Example in medicine: methotrexate competes with a normal substrate for the enzyme dihydrofolate reductase and is used in cancer treatment.

What is non-competitive inhibition?

A non-competitive inhibitor binds to a different part of the enzyme, called the allosteric site, away from the active site. Binding changes the shape of the enzyme, including the shape of the active site, so that the substrate can no longer bind properly or the reaction cannot occur. The inhibitor does not resemble the substrate.

• Increasing the substrate concentration does not overcome the effect, because the inhibitor is not competing for the active site. Some enzyme molecules are put out of action, and so V_max is lowered.
• Examples: cyanide inhibits cytochrome c oxidase in the electron transport chain, and heavy metals such as mercury and lead bind to enzymes and damage them.

Allosteric regulation is the natural control of enzymes by molecules that bind to allosteric sites. Activators stabilize the active form of the enzyme, and inhibitors stabilize the inactive form. End-product (feedback) inhibition, such as the inhibition of phosphofructokinase by ATP in glycolysis, often works this way. It allows the cell to adjust the rate of pathways quickly.

Competitive and non-competitive inhibition compared

What are cofactors and coenzymes?

Many enzymes are not active alone, and need a non-protein helper called a cofactor. An enzyme without its cofactor is an apoenzyme (inactive), and with its cofactor it is a holoenzyme (active).

• Inorganic cofactors (metal ions): Zn²⁺ in carbonic anhydrase and alcohol dehydrogenase, Mg²⁺ in many enzymes that use ATP, Fe²⁺ in catalase and cytochromes, and Cu²⁺ and Mn²⁺ in others. They may help to bind the substrate or take part in the reaction. A diet that lacks these minerals reduces the activity of such enzymes.
• Coenzymes (organic molecules): small organic molecules that carry atoms or electrons from one reaction to another. Many are made from vitamins: NAD⁺ and NADP⁺ (from niacin, vitamin B3) carry hydrogen and electrons in respiration and photosynthesis, FAD (from riboflavin, B2) carries hydrogen, and coenzyme A (from pantothenic acid, B5) carries acetyl groups. ATP can also be viewed as a coenzyme.
• Prosthetic groups: cofactors that are permanently attached to the enzyme, such as the heme group in catalase.

Because many coenzymes come from vitamins, a lack of a vitamin may lead to enzyme deficiency. For example, a lack of niacin causes pellagra and a lack of thiamin (B1) causes beriberi.

How are inhibitors used in medicine and nature?

• Antibiotics: penicillin inhibits an enzyme that builds the bacterial cell wall (it binds irreversibly). Sulfonamides are competitive inhibitors of an enzyme in folic acid synthesis, because they resemble the substrate PABA.
• Blood pressure drugs: ACE inhibitors, such as ramipril, inhibit angiotensin-converting enzyme.
• Cholesterol: statins inhibit HMG-CoA reductase, an enzyme in the synthesis of cholesterol.
• Pain relief: aspirin irreversibly inhibits cyclooxygenase, reducing the formation of prostaglandins.
• Poisons: nerve gases such as sarin inhibit acetylcholinesterase, so acetylcholine accumulates at synapses; organophosphate insecticides act in a similar way.
• Natural control: end-product inhibition regulates metabolism.

Worked example: overcoming competitive inhibition

An enzyme has a V_max of 100 units. Without inhibitor, its K_m is 2 mmol/L. A competitive inhibitor makes the apparent K_m 6 mmol/L. Use the Michaelis-Menten equation, rate = V_max × [S] ÷ (K_m + [S]), to compare rates.

1. At 2 mmol/L: no inhibitor: 100 × 2 ÷ (2 + 2) = 50. With inhibitor: 100 × 2 ÷ (6 + 2) = 25. So the inhibited rate is 50 percent of the normal rate.
2. At 20 mmol/L: no inhibitor: 100 × 20 ÷ 22 = 90.9. With inhibitor: 100 × 20 ÷ 26 = 76.9, which is 85 percent.
3. At 100 mmol/L: 100 × 100 ÷ 102 = 98.0, and 100 × 100 ÷ 106 = 94.3, which is 96 percent.

The inhibition becomes weaker as the substrate concentration increases, as it should for a competitive inhibitor, and the rate approaches the same V_max. For a non-competitive inhibitor that reduced V_max to 60, the inhibited rate would be about 60 percent of the normal rate at every substrate concentration, which would not improve with extra substrate.

How were these concepts developed?

Leonor Michaelis and Maud Menten described enzyme kinetics in 1913. Competitive inhibition was studied by Donald Woods and Paul Fildes in 1940, who showed that sulfonamides compete with PABA in bacteria, an early example of rational drug design. Jacques Monod, Jean-Pierre Changeux and François Jacob introduced the idea of allosteric regulation in the early 1960s. Arthur Harden and William Young found in 1906 that a heat-stable, small molecule is needed with the enzymes of fermentation, which led to the concept of coenzymes.

Which mistakes are common?

Mistake 1: Saying that a competitive inhibitor changes the shape of the enzyme

It blocks the active site. A non-competitive inhibitor changes the shape of the enzyme.

Mistake 2: Thinking that a competitive inhibitor is permanent

It is reversible. More substrate can displace it.

Mistake 3: Confusing cofactor and coenzyme

Cofactors can be metal ions or organic molecules. Coenzymes are organic cofactors, usually derived from vitamins.

Mistake 4: Saying that inhibition always harms the cell

Inhibition is also a natural way of controlling metabolism, as in feedback inhibition.