Denaturation and the chemistry of enzyme action

What denaturation is

A protein works only when it is folded in its own shape. Denaturation is the loss of this shape. The secondary, tertiary and quaternary structures are destroyed. The primary structure is not: no peptide bond breaks, and the sequence of residues stays the same.

Several things denature proteins:

• Heat. Higher temperature shakes the hydrogen bonds and the hydrophobic contacts apart. This is why an egg white sets when it is boiled.
• Acids and bases. A change of pH changes the charges on the side chains, and the ionic attractions break.
• Urea or guanidine at high concentration. They disturb the hydrogen bonds and the water structure that holds the hydrophobic core together.
• Organic solvents and detergents. They dissolve the non-polar core.
• Heavy metal ions, such as $\text{Hg}^{2+}$ and $\text{Pb}^{2+}$. They bind to $\text{-}\text{SH}$ and $\text{-}\text{COO}^{-}$ groups. This is why mercury and lead salts are poisons.

A denatured protein is often less soluble. The exposed non-polar parts stick together, and the protein coagulates. Milk curdles when lactic acid brings the pH down to about 4.6, the pI of casein, the main milk protein. At the pI the protein has no net charge and clumps together.

Denaturation and hydrolysis are different. Denaturation unfolds the chain. Hydrolysis cuts the peptide bonds. Hydrolysis needs a strong acid and heat for many hours, or an enzyme.

Some denaturation can be undone. Ribonuclease A was unfolded with urea and a reducing agent. When both were removed slowly, the enzyme folded again and worked. The experiment, by Anfinsen, showed that the sequence holds the information for the fold. A boiled egg does not become raw again, because the proteins clump irreversibly.

Enzymes

Enzymes are the catalysts of living cells. Nearly all are proteins. They have five features:

• Efficiency. They raise the rate of a reaction by factors of a million or more.
• Specificity. One enzyme works on one substrate, or on a small group of similar ones. Urease acts on urea only.
• Mild conditions. They work in water, near 37 °C and near neutral pH. Pepsin works best near pH 2 in the stomach. Trypsin works best near pH 8 in the intestine.
• They do not change the equilibrium. They only make it reached sooner. They are not used up.
• Control. Their activity can be switched on and off by other molecules.

The names usually end in -ase. Maltase acts on maltose, lactase on lactose, and zymase in yeast converts glucose to ethanol. Many enzymes need a small helper, a coenzyme. Several vitamins become coenzymes, which we meet in the next course.

How an enzyme works

The substrate binds in a pocket on the enzyme, the active site. Two models describe the fit:

• Lock and key (Fischer): the substrate fits the site as a key fits a lock.
• Induced fit (Koshland): the enzyme changes shape a little when the substrate arrives, and grips it better.

Binding is only the start. Bonds in the substrate must break and form. This needs energy, the activation energy. An enzyme gives the reaction a new path with a lower barrier.

The enzyme does this by holding the substrate in the right place and by stabilising the transition state, the unstable arrangement at the top of the barrier. Amino acid side chains in the active site act as acids, bases or nucleophiles.

A well-studied example is chymotrypsin, which cuts peptide bonds next to aromatic residues. Three residues work together, the catalytic triad: serine, histidine and aspartate (Ser 195, His 57 and Asp 102 in the usual numbering). The histidine takes a proton from the serine $\text{-}\text{OH}$ group. The serine oxygen then attacks the carbonyl carbon of the peptide bond and forms a covalent bond with it. Water later completes the cut and frees the enzyme.

Putting a number on it

If the frequency factor does not change, the Arrhenius law gives the ratio of the rates:

Take a reduction of the activation energy by 30 kJ/mol at 310 K. Then $\Delta E_a/RT = 30000/(8.314 \times 310) = 11.64$, and the ratio is $e^{11.64} \approx 1.1 \times 10^5$. A 30 kJ/mol change in the barrier makes the reaction a hundred thousand times faster.

Temperature and pH

The rate of an enzyme reaction rises as the temperature rises, as the Arrhenius law says. But the enzyme starts to unfold. Above a certain temperature the loss of folded enzyme wins, and the activity falls. This gives an optimum temperature, near 37 °C for most human enzymes. Similarly, an enzyme has an optimum pH, because the charges on the active-site groups must be right.

Raise the activation energy. The Arrhenius curve (grey) gets steeper, and the peak moves up. Now lower the temperature at which half the enzyme is unfolded. The peak moves left and gets smaller.

Inhibitors

An inhibitor is a molecule that slows an enzyme. A competitive inhibitor looks like the substrate and blocks the active site. Many drugs work in this way. Ethanol is given in the treatment of methanol poisoning, because it competes with methanol for the same enzyme, alcohol dehydrogenase. Other inhibitors bind elsewhere and change the shape of the enzyme.

Practise with new numbers

Try it in Python

The program works out how much faster an enzyme makes a reaction. It then scans the temperature to find the optimum of an enzyme that is half unfolded at 328 K.

Common mistakes

• Saying that denaturation breaks peptide bonds. It breaks only the weak interactions and the disulphide bridges, and the sequence stays.
• Thinking an enzyme changes the position of equilibrium. It speeds the way to equilibrium in both directions.
• Thinking a higher temperature always means a faster enzyme reaction. Above the optimum, the enzyme unfolds.
• Mixing up the lock-and-key and the induced-fit models. In the second, the active site adjusts.

Remember these

• Denaturation destroys the secondary, tertiary and quaternary structures. The primary structure stays.
• Heat, pH change, urea, solvents and heavy metal ions denature. Milk curdles at the pI of casein, about pH 4.6.
• An enzyme lowers the activation energy and does not change the equilibrium. It is specific and works in mild conditions.
• Rate ratio $= e^{\Delta E_a/RT}$. A 30 kJ/mol drop at 310 K gives a factor of about $10^5$.
• Activity has an optimum temperature and an optimum pH.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. After a protein is denatured, is its molar mass the same? Is its amino acid sequence the same?

2. Why does an enzyme not change the equilibrium constant of a reaction?

3. Why is there an optimum temperature for an enzyme?

4. Why is mercury(II) chloride poisonous?

References

• Nelson and Cox, Lehninger Principles of Biochemistry, 8th edition, Chapter 4 (denaturation, Anfinsen's experiment) and Chapter 6 (enzymes, the catalytic triad).
• NCERT Chemistry for Class 12, Part II, the chapter Biomolecules (denaturation of proteins, enzymes).