Enzyme Function

Question 1

Why do enzymes work?

Answer 1

Fatty acid amide hydrolases:

• breaks down neuroactive signals
• part of the endocannabinoid signalling system. Inhibitors promote analgesia, and can prevent effects of chronic stress in mice (but it doesn’t appear to work in humans).
• if you could inhibit the enzyme, the concentration of the neuroactive molecule should go up, resulting in reduction in pain sensation

Question 2

Key ideas from last year

Answer 2

1. Any reaction has a “transition state”, a high energy point on the path from the starting molecules to the products.
2. The energy barrier between the starting point and the transition state determines the rate of the reaction (in standard conditions).
3. Enzymes act by reducing the energy barrier, and so increasing the proportion of molecules that have the energy to access the transition state.

Question 3

Protein structure is critical to function

Answer 3

Principles for enzyme catalysis:

Aim is the stabilisation of transition state (lowers activity barrier)

• Near-attack conformations
• Covalent catalysis
• Acid-base catalysis
• Low barrier hydrogen bonds
• Metal ion catalysis
• “Induced fit”
• Hydrophobic “binding pockets”

Question 4

Induced fit

Answer 4

The folding of a protein allows enzymes to stabilise the transition state indirectly.

The protein can fold so that there are some unfavourable elements left in the folded protein.

Binding of substrate, and especially of the transition state, alters the protein conformation so that the protein is more stable.

This reduces the energy required to access the transition state.

Question 5

Metal ions in catalysis

Answer 5

Metal ions generally act in one of three manners to drive catalysis:

• Redox chemistry – see BIO2091.
• Binding of substrates.
• Electrostatically stabilising or shielding negative charges.

Metal ions can also hold parts of the protein together (like a disulfide bond, but metal ions can occur in the cytoplasm whereas disulfide bonds cannot be maintained in the reducing environment of the cytoplasm)

Question 6

Electrostatic stabilising of charges

Answer 6

Metal ions commonly act to stabilise negative charges in the transition state.

This stabilises the transition state, by reducing the energy penalty to form these charges (significantly).
The rate should then increase.

Question 7

Activating water

Answer 7

Metal ions commonly act to activate water, allowing the formation of highly reactive OH- ions.

Example: Ribonuclease D.

Question 8

Acid-base pairs

Answer 8

Many reactions are driven by movement of hydrogen ions.

Requires that suitable donors and acceptors of protons are available – rare at neutral pH.

Proteins are very capable of generating side chains that can readily donate protons (Brønsted acid) or accept them (Brønsted base).

Question 9

Summary

Answer 9

Protein structure is critical to function.

Mechanisms for increasing the rates of reactions include:

Covalent catalysis, where the enzyme makes a covalent intermediate in the reaction.

Stabilising charged (or unusually hydrophobic) intermediates using other parts of the active site.

Use of metal ion point charges to drive catalysis.

Acid and base groups are used by enzymes to facilitate proton movements.