Kinetics and Catalysis

Question 1

Importance of enzyme science

Answer 1

• Metabolic engineering
• Biosensors
• Drug discovery
• Diagnostics
• Systems biology
• Synthetic biology

Question 2

Chemical kinetics

Answer 2

The general reaction may have multiple substrates or products

There may be multiple copies of any of these molecules in the reaction mechanism

Examples:
1. Isomerases:
take the atoms in a molecule and rearrange them in some manner. These enzymes usually have one substrate and one product

2. Transferases:
   take some atoms from one molecule and add them to another. These enzymes usually have two substrates and two products.

• Kinases: a type of Transferase
  These molecules transfer a phosphate from ATP to a target molecule (example – which I studied in my PhD - phosphorylates a tyrosine in a target protein). Approximately 3.5% of our genome is given over to kinases and enzymes that catalyse the reverse reaction, and these make up about 20% of targets of currently marketed drugs.
• 60% of enzymes are 2 substrate-2 product varieties

Question 3

The four other main classes of enzymes

Answer 3

Hydrolases, lyases, oxidoreductases and ligases.

Question 4

Rate equations

Answer 4

For the general reaction:
aA + bB + … + zZ —> P
The rate is:
Rate = k [A]a [B]b … [Z]z
This applies if all the reactants mix simultaneously. In practice, only two or at most three will ever mix at the same time.
The “order” of the reaction is given by the number of molecules mixing.

Question 5

Rates of reactions

Answer 5

The rate can be described as:

v = -d[A]/dt = d[P]/dt = k[A]

This equation can be altered to allow experimental determination of k

Question 6

Measuring reactions

Answer 6

1. Spectrophotometry (UV/VIS)
2. Fluorescence:
   Many molecules are fluorescent, so their quantity can be followed.
   e.g. Tryptophan in proteins changes fluorescence when the protein is ligand bound.
   More complicated schemes (time resolved fluorescence, fluorescence polarisation) offer better sensitivity.
3. Luminescence
4. FTIR spectroscopy
5. Synchrotron radiation

Other physical principles may be used:
Radioactivity (frowned on nowadays)
Mass spectrometry
Capillary electrophoresis
Agarose gels (common for nucleotide experiments)
HPLC
Anything that lets you make an accurate enough measurement

Most of these methods have the drawback that they can only be used for stopped reactions. This is why UV-Vis spectroscopy continues to be so popular – it allows continuous assays.

Question 7

Main types of enzyme measurements

Answer 7

Simple, continuous assay:
This is a reaction where one of the products can be directly measured

The reaction has been measured here making sure that there is a stable baseline.
The rate measured is only that in the first minute or so that there is a good measurement.
As the reaction proceeds, the rate drops away – this is why only the initial rate is measured.

Stopped assay:
In this experiment, the assay was stopped by heating the reactants after a set time to quench the reaction. The reaction is measuring phosphate release by an enzyme. The phosphate is detected using malachite green in strong acid, and measured at 635 nm. This means that only a single time point is measured.
This is very laborious, and so continuous assays are preferred.

Coupled assay:
In a coupled assay, a second enzyme is added to the reaction, generally to provide a measurable product that can be followed. Often, coupled assays are also used in stopped reactions where there is no good assay for either substrate or product of the desired reaction. The product of the reaction of interest will now be an intermediate in the measured reaction. As a result, there is delay after adding the enzyme, before the reaction achieves a full rate. This “lag phase” occurs whilst the concentration of the intermediate reaches a steady state (where it is used up as fast as it is produced). The lag phase length will depend on the relative amounts and rates of the two enzymes. With a continuous assay, it is easy enough to tune the lag phase to a sensible point. In a coupled assay, of course, it is all rather more complicated…

Question 8

Enzyme kinetics and the Michaelis-Menten equation

Answer 8

Key to understanding this is the concept that the enzyme-substrate complex reaches a steady state. This means that more ES complex forms at the same rate that it forms product or releases the substrate.
(𝑑[𝑆])/𝑑𝑡=0
This is the steady state approximation.

Question 9

Summary

Answer 9

We can measure enzymes using any available method.

Continuous assays are ideal; coupling is used to make this possible.

Experiments need building up carefully.