Enzymes are proteins that catalyse virtually all the biochemical reactions happening in the body, working at body temperature (37°C) and atmospheric pressure, and increasing the rate of reaction by at least a million-fold. They provide an alternative pathway for the reaction with a lower activation energy, just like non-biological catalysts.
Enzymes are highly specific. In the ‘lock and key‘ model of enzyme catalysis, the substrate molecule binds to the active site of the enzyme (usually a cleft or groove on the surface of the enzyme) forming an enzyme-substrate complex. The substrate is held in place by a variety of intermolecular bonds, ionic bonds, and hydrophobic interactions. The substrate is converted to the product and the product is then released.
Clearly this is a simplified picture and it is more likely that the conformations of both the enzyme and substrate are modified by substrate binding (this is known as the ‘induced fit‘ model). The stress weakens key bonds in the substrate, lowering the activation energy for the reaction.
The example below shows the enzyme chymotrypsin hydrolysing a peptide link and catalysing the breakdown of a protein:
Most biochemical reactions involve two or more different substrates reacting together e.g. forming a dipeptide from two amino acids. The binding of two or more substrates to the active site in the proper position and orientation encourages formation of the transition state which then moves quickly to formation of the products.
E + S ⇌ E-S ⇌ E-P ⇾ E + P
We can illustrate this on an energy profile diagram:
Effect of pH and temp changes on active site
Enzymes are particularly sensitive to changes in pH and temperature.
- pH
Changes in pH will affect the degree of ionisation of many of the R-groups of amino acids in the polypeptide chains of the enzyme e.g. -NH2 / -NH3+ in arginine, histidine and lysine and -COOH / -COO– in aspartic acid and glutamic acid. This has implications for any ionic interactions between the active site and substrate and for H+ ion transfers.
The optimum pH for many enzymes is around 7 but obviously enzymes working in the stomach will be most effective at a much lower pH (e.g. pepsin has optimal activity around pH 1.5) and the pH for those working in the small intestine will be on the alkaline side.
- Temperature
Enzyme activity increases with rising temperature until around 40°C (more enzyme and substrate collisions exceed activation energy) but then activity decreases sharply.
The weak intermolecular bonds that hold the tertiary structure of a protein together are easily broken by increased vibration at higher temperatures. The active site loses its 3D shape and the enzyme is eventually denatured – this is irreversible.
Enzyme kinetics
The rate of an enzyme catalysed reaction depends on a number of factors – whether temperature and pH are optimal, the affinity of the substrate for the enzyme’s active site and the concentration of both the enzyme and the substrate.
The concentration of enzyme is always very low compared with the concentration of substrate so reaction is always first order with respect to the enzyme.
However if we look at the graph below we can see that the order of reaction with respect to the substrate changes depending on the concentration …
Inhibitors
Competitive inhibitors compete with the substrate to occupy the active site of an enzyme. They often have a similar shape to the substrate and can form intermolecular bonds with the active site. Significantly increasing the concentration of the substrate may allow it to outcompete the inhibitor.
Some inhibitors bind to part of the enzyme that is not the active site, changing the shape of enzyme so that either the substrate cannot enter the active site, or preventing the substrate form forming key bonds with the active site. Increasing [S] will have no effect on the rate of the reaction.
Many pharmacological drugs are enzyme inhibitors. For example, aspirin inhibits an enzyme that plays a key role in the production of pro-inflammatory molecules in the body, hence its use to reduce pain and inflammation.
Practice questions
The protease enzymes hydrolyse peptide bonds in polypeptide chain substrates adjacent to hydrophobic amino acids, such as tryptophan and phenylalanine. The amino acid adjacent to the peptide bond to be cleaved is inserted into a pocket at the active site of the enzyme.
(a) Given that the general formula for an amino acid is H2N-CH(R)-COOH, suggest a structure for the amino acid phenylalanine.
(b) Identify the peptide bond that is targeted by the enzyme for hydrolysis.
(c) The polypeptide chain is also held in place by interactions between the amino acid glutamine and the enzyme.
(i) Suggest what type of interaction is most likely.
(ii) Explain the effect of lowering the pH on the enzyme’s activity.
(d) This protease enzyme is inhibited by the molecule shown below:
(i) What is an inhibitor?
(ii) Explain how this molecule affects the function of the enzyme.
Answers
(a)
(b)
(c) (i) hydrogen bonds could form between the -NH2 group of glutamine and the enzyme.
(ii) Lowering the pH means the environment would be more acidic. The -NH2 group on the glutamine amino acid residue in the polypeptide chain would behave as a base, accepting a H+ to become -NH3+ and it would no longer be able to form hydrogen bonds with the enzyme. This would affect the ability of the polypeptide substrate to bind to the active site of the enzyme, reducing its activity as a catalyst.
(d) (i) An inhibitor is a molecule that blocks or preferentially binds to the active site of an enzyme preventing the substrate from binding.
(ii) This inhibitor molecule has a similar shape to the substrate, with a hydrophobic group that can bind to the pocket in the active site of the enzyme, just like the substrate. It also has a glutamine R-group in the same place as the substrate which can make hydrogen bonds to the enzyme, holding the inhibitor in place.
The enzyme catalyses the hydrolysis of the peptide link adjacent to the phenylalanine amino acid residue in the substrate, after which the product molecules leave the active site and the enzyme can continue to work. The inhibitor molecule does not have an amide / peptide bond in the same place so once it has bound to the active site it will stay, preventing the enzyme from working.