Team:Imperial College London/Drylab/Enzyme/Analysis

Assumptions

In the standard model of enzymatic reaction, 3 sets of assumptions are made.

Enzymatic assumptions:

• The enzyme is specific only for the substrate and not for any other chemicals.

• Only one enzyme, our enzyme of interest is present and participating in the reaction.

• There is negligible formation of product without the enzyme.

• The rate of enzymatic activity remains constant over time because there is:
  • no co-operativity of the system. Binding of substrate to one enzyme binding site doesn't influence the affinity or activity of an adjacent site.
  • no allosteric regulations from either the product or the substrate.
  • no product inhibition of the enzyme.

• The enzymatic reaction can be modelled by the following set of reactions (in particular the catalysed reaction is irreversible)

ie:

Degradation assumptions:

• All proteins are very stable and thus their degradation can be neglected over the course of the experiments
• If there is no creation of substrate during the experiment (which is the most common case), we thus have [S]0=[S]+[ES]+[P] at all time
• Likewise if there is no creation of enzyme during the experiment (which is the most common case), we have [E₀] =[E]+[ES] at all time

Law of mass action assumptions:

• Free diffusion ; Free unrestricted thermodynamically driven random molecular motion
• The reagents are in thermal equilibrium at a constant absolute temperature
• The reagents are well mixed. [2]

Michaelis-Menten assumption:

• The Michaelis-Menten (MM) assumption is a simplifying assumption that is commonly made with enzymatic reactions. It says that since substrate binding is very fast compared to catalysis, the enzyme complex ES is always at quasi-steady state, ie: , during the time of the experiment.

• It is straightforward to prove that combining the MM assumption with the conservation equation [E₀]=[E]+[ES] yields a new form for the fourth equation , where KM is the Michaelis-Menten constant.

• From the above assumptions, we know that

Properties of a Standard Enzymatic Reaction

1) When substrate concentration is large, the enzyme concentration is the limiting factor, hence rate of reaction is directly proportional to [E].

2) At very low [E], the reaction rate measured will be negligible as very low amounts of product will be produced

3) Increasing k1 and k3 values will increase the product synthesis rate

4) When substrate concentration is large, the enzyme concentration is the limiting factor, hence rate of reaction is directly proportional to [E].

5) At very low [E], the reaction rate measured will be negligible as very low amounts of product will be produced

6) Steady state approximation will only be valid when [S₀]>>[E₀]

The actual model...