Enzyme kinetics describe the mathematical properties of how enzymes behave, but what they don't do is describe the mechanistic basis, that is what happens at a molecular level during the course of an enzyme catalyzed reaction.
Understanding what happens at a molecular level in an enzyme can be important if an enzyme is clinically important. Many drugs are enzyme inhibitors or otherwise have a mechanism of action that involves enzyme catalysis, thus studying the mechanism of enzymes can be an important aspect of rational drug design.
Enzymes as proteins can be studied using many of the same techniques that can be used to study proteins generally. Their structures can be derived using techniques such as X-ray crystallography or nuclear magnetic resonance, and their protein and DNA sequences can be derived from mass spectrometry or DNA sequencing respectively. However there are some techniques that are used specifically for enzymes.
Affinity Tags
A typical experiment for studying an enzyme would go something like this: A scientist would hypothesize, based on the enzyme structure, or sequence conservation, that a particular amino acid has a particular function in the enzyme catalysis. For example, a scientist might hypothesize that a serine reside in an active site of an enzyme donates a hydrogen ion to create an acidic environment within the active site, and this allows an acid catalyzed reaction to complete faster.
An affinity tag is a compound that can bind specifically and with high affinity (often using a covalent bond) to a specific amino acid residue. By binding to a specific amino acid, the affinity tag is able to stop whatever function that amino acid carried out in the enzyme catalysis from happening. By disabling a selected amino acid, then studying the enzyme with that disabled amino acid in comparison to the regular enzyme, the contribution of that amino acid to the enzyme function can be derived.
An example of an affinity tag is organophosphate (Figure 1). Organophosphate molecules bind irreversibly to the serine amino acid, thus disabling it. It does so to the SER residue of the acetylcholinesterase enzyme, which is an enzyme that breaks down acetylcholine, an important neurotransmitter. Thus indicating that serine is of vital importance to this enzyme function.
Figure 1: An organophosphate, such as sarin, forms a covalent bond with serine amino acids. This has the effect of eliminating any function of the serine amino acid in the enzyme. This affinity tag is useful to determine that serine has a key role in the function of the enzyme, and similar affinity tags can be used to target other amino acids. However one of the enzymes that relies on a serine amino acid is acetylcholinesterase, an enzyme involved in the correct functioning of the nerves, thus sarin, and similar organophosphate molecules are extremely neurotoxic.
Affinity tags have numerous problems. Firstly an appropriate affinity tag needs to be identified for the amino acid being investigated. Secondly affinity tags are only specific for the amino acid, not for the position of the amino acid. Thirdly, affinity tags, as powerful enzyme inhibitors can be extremely toxic. Organophosphates are better known for their use as nerve gases for example. Because of these shortcomings, better ways of studying enzymes are used, such as genetic engineering.
Genetic Engineering
Genetic engineering is a technique that uses molecular biology techniques to precisely change the DNA sequence of a gene, and thus change the resulting protein sequence. As proteins, including enzymes can be produced from a given DNA sequence relatively simply from bacterial cultures in the laboratory, this means it is relatively simple to produce genetically engineered enzymes.
Genetic engineering can more precisely modify the composition of an enzyme based on the hypothesis under investigation. Firstly unlike an affinity tag that will bind to any amino acid residues of a particular type, genetic engineering can change a single amino acid. Additionally, the change can replace a given amino acid with a similar one, with a difference in the particular functional effect under investigation. If the -OH group in a tyrosine residue is speculated to have a role in the enzyme function, this residue can be switched with a phenylalanine, which has the same benzene ring structure as tyrosine, but lacks the -OH group (Figure 2).
Figure 2: Serine (left) and alanine (right) are very similar amino acids, differing only in the -OH group that serine has which alanine does not have. In a genetic engineering experiment, the serine amino acid (or rather a specific serine amino acid at a particular part of the enzyme) can be changed, by modifying the DNA encoding the enzyme, to an alanine amino acid. This engineered enzyme can then be produced, and assayed, with its results compared to those of the original enzyme. Depending what effects on enzyme function are observed, the role of that amino acid in the enzyme function can be predicted.
Other amino acids can have similar changes made to them, depending on the hypothesis being tested. This makes genetic engineering a powerful tool for studying enzymes. Very precise changes can be made to the amino acid contribution to test a defined hypothesis precisely.
