Dear Sir. Concerning your issue about the about the enzyme concentration used for Km determination. Michaelis constants have been determined for many of the commonly used enzymes. The size of Km tells us several things about a particular enzyme. A small Km indicates that the enzyme requires only a small amount of substrate to become saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations. A large Km indicates the need for high substrate concentrations to achieve maximum reaction velocity.The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently assumed to be enzyme's natural substrate, though this is not true for all enzymes. In order to study the effect of increasing the enzyme concentration upon the reaction rate, the substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate concentration. Any change in the amount of product formed over a specified period of time will be dependent upon the level of enzyme present. The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship between activity and concentration is affected by many factors such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the reaction is zero order. For more details, I think the following below link and the attached file may help you in your analysis:

http://www.worthington-biochem.com/introbiochem/Enzymes.pdf

Thanks

Hi there,

There are actually 2 issues:

One of the condition for using Michaelis Menten equation is that enzyme concentration is by far less than the substrate concentration. Usually there is at least 3 orders of magnitude between the 2 concentrations.

Measuring initial velocities (as Michaelis Menten equation is valid only for initial velocities)  implies that during the laps of time considered product is formed linearly versus time. So for the higher concentration of substrate considered, the concentration of enzyme has to be adjusted so that initial velocity can be measured. Once this enzyme concentration determined with the higher substrate concentration, any other kinetics ran on the same laps of time with the same concentration of enzyme but with lower substrate concentration will automatically lead to determination of the associated initial velocity.

A suitable enzyme concentration should be determined by experimentation. For low substrate concentrations (relative to the Km), depletion of the substrate causes the reaction to slow down more than at higher substrate concentration, so a low enzyme concentration is needed to maintain the initial rate long enough for the initial rate measurement to be made.

A good way to get started is to run a matrix of different substrate and enzyme concentrations. This allows you to get a rough estimate of the Km and also identify a suitable enzyme concentration to use for more detailed experiments.