Hi there,

Enzyme kinetics is done by following the evolution in time of the content of the reaction sample: in general one follow the reaction by detecting and quantifying either the appearance of the reaction product or disappearance of the reaction substrate (depending on what is the easiest). Enzyme kinetics will vary according to different parameters including t°, pH, salt, substrate(s) concentration and enzyme concentration. In the enzyme assay, parameters are set in order to follow the enzyme kinetics during initial velocity window (period of time during which the reaction rate is constant, ie appearance of the product or disappearance of the substrate is linear towards time).

Thanks Dominique.

It was very helpful. It means enzyme kinetics contains several parameters but enzyme assay contains one or few and it is all about to check the reaction where the enzyme is stable.

I'll expand on Dominique's answer. When setting up an enzyme assay to study inhibition, it is worthwhile to use enzyme kinetics to measure the Michaelis constant (Km) for the enzyme's substrate, or for each of the substrates if it is a multisubstrate enzyme. The reason for doing this is so that you can understand the relationship between the substrate concentration(s) in the assay and the sensitivity of the assay to competitive inhibition.

For example, consider a single-substrate enzyme with a Km of 10 µM. You might like to put 100 µM substrate (10X Km) in the enzyme assay to get a large signal while keeping the reaction running at the initial rate. If you do this, the ratio of the IC50 to the dissociation constant of the inhibitor (Ki) (assuming an inhibitor in rapid equilibrium binding with the enzyme) IC50/Ki = 1 + [S]/Km = 11.

In contrast, if you use 10 µM substrate in the reaction and still keep the reaction at the initial rate, then IC50/Ki = 2. So the IC50 of the inhibitor will be much higher in the first case than in the second case, even though the affinity of the inhibitor is the same.

The math gets more complicated for multi-substrate enzymes, but the concept is the same.

Notice the importance of making sure the reaction runs at its initial rate during the time when you are measuring inhibition. The reaction will slow down as the substrate is used up and the product(s) accumulate(s), but if you measure inhibition after the reaction starts to slow down, the inhibitor will appear less potent than it really is (IC50 increases).

A further complication that can arise is that an inhibitor can sometimes show time dependence, becoming more potent with time of exposure to the enzyme. This can be seen by following the reaction progress over time at various concentrations of inhibitor.

it is very informative...thanks shapiro and liger.

Enzyme assay is a parameter to evaluate enzyme kinetics parameters

If you want to perform inhibition assay, you may do a kinetic assay using the inhibitor against different concentrations of substrate. And then, do a linear regression, can be a Lineweaver Burk plot.

Very conceptual n very informative answer Mr Adam..............Thanks