[parkersa]

I want to compare two enzymes possessing the same activity.  I have a nice calorometric assay with change in color to yellow I can read on a plate reader over time.  I want to test a particular substate, which is very hard to make.  I cannot make enough of the substrate to feasibly do full kinetics.  One of the enzymes in pilot studies also appears not to be as active on this substrate, and to see a reliable color change I must use about 8x the concentration of the other enzyme (0.46uM vs 3.8uM).  Because substrate is limited, I can only use a max of 3.5x the enzyme concentration.  So in lieu of full kinetics, am thinking of presenting the data as moles of substrate converted per second per uM of enzyme, and showing time curves.

Does this seem acceptable within the limitations I present, or is there a better way to present data when substrate is limited?

Is there a rule of thumb about the MINIMUM substrate to enzyme ratio I should abide by?

What would you see as the limitations of such data that should be recognized as such in a manuscript?

Thank you,

[jdy07]

Don't know if this is too late for you, but thought I might try to answer anyway. I assume you are asking if you can do a steady-state activity plot (e.g. Michaelis-Menten plot). Generally, in order to do these type of plots you need to be in pseudo first order conditions or around 10X substrate over enzyme. The best you may be able to do is to determine an apparent kcat from the data you have and report this as a lower limit on the actual kcat. Another option if your assay is really sensitive is to have the enzyme in 10X excess over the substrate since either the substrate OR the enzyme can be in excess to use the pseudo first order approximation. Hope this helps.