I recognize that in a standard enzyme kinetics assay the protein concentration should be negligible, however I have recently come across many assays in which researchers will utilize really high concentrations of protein and substrate. For example 1uM of protein vs. 100uM of substrate. The stoichiometry is 1:100, but the concentrations are significantly higher most other assays I have ever seen. The other issue is that the researchers are trying to determine what substrate preferences the enzyme has without a full on kinetic analysis, but I would guess that if you are assaying an enzyme at such high concentrations you might get all sorts of non-specific enzyme binding and activity.
The requirement for steady-state enzyme kinetic analysis is that the free substrate concentration is essentially identical to the total substrate concentration. This is certain to be true when the enzyme concentration is far below the substrate concentration. There is not a requirement that the enzyme concentration be "negligible," only that the fraction of the substrate bound to the enzyme is negligible.
In the case you mentioned, the enzyme concentration was 1% of the substrate concentration. Assuming that the substrate concentration is high enough to saturate the enzyme ([S]>>Km), then only 1% of the substrate will be bound (less if the substrate concentration is not saturating), which is satisfactory for the purpose of steady-state kinetic analysis.
The problem with a "high" enzyme concentration may be that the reaction goes so fast that the initial rate can't be measured. However, if the enzyme has a low rate of catalysis with a particular substrate, the high enzyme concentration may be necessary to get a sufficiently high rate to measure. As long as the conditions for steady-state kinetic analysis are met, this could be an acceptable setup for the experiment.
Trying to draw conclusions about substrate preferences without a thorough kinetic analysis, including measurements of kcat and Km (or Kms if it is a multisubstrate reaction) is unwise.
The requirement for steady-state enzyme kinetic analysis is that the free substrate concentration is essentially identical to the total substrate concentration. This is certain to be true when the enzyme concentration is far below the substrate concentration. There is not a requirement that the enzyme concentration be "negligible," only that the fraction of the substrate bound to the enzyme is negligible.
In the case you mentioned, the enzyme concentration was 1% of the substrate concentration. Assuming that the substrate concentration is high enough to saturate the enzyme ([S]>>Km), then only 1% of the substrate will be bound (less if the substrate concentration is not saturating), which is satisfactory for the purpose of steady-state kinetic analysis.
The problem with a "high" enzyme concentration may be that the reaction goes so fast that the initial rate can't be measured. However, if the enzyme has a low rate of catalysis with a particular substrate, the high enzyme concentration may be necessary to get a sufficiently high rate to measure. As long as the conditions for steady-state kinetic analysis are met, this could be an acceptable setup for the experiment.
Trying to draw conclusions about substrate preferences without a thorough kinetic analysis, including measurements of kcat and Km (or Kms if it is a multisubstrate reaction) is unwise.
Every enzyme has a fixed number of binding site availability for a particular ligand, i.e., the binding stoichiometry has a definite range. If excessive binding occurs Kcat value changes hence the enzyme activity changes.
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