I'm trying to establish the Km for different substrates for a specific enzyme, however, at every attempt a new doubt arises. This is a new field of knowledge to me, so any suggestion will be helpful.
So far I am following these premisses:
- at least 6 substrate concentrations, sometimes 7 (from 1 mM to ~ 0.015 mM) ;
- the substrate consumption should be less than 10% ( so I am testing several amounts of enzyme for each substrate and using the amount that is enough to be measured and also consumes less than 10% of the substrate, even in the lowest concentrations);
- Results of the Km and Vmax are obtained by nonlinear regression of the Michaelis-Menten curve using GraphPad Prism software.
The analysis is performed by the formation of the phosphate in the reaction (molybdate/malachite green-based assay).
Some of the problems I'm facing:
1) for some substrates I couldn't get an M-M-like curve, even when trying different amounts of enzyme or substrate. Then, I believe that the data are not adequate to perform Km and Vmax calculations. I've noticed that with these substrates the consumption is lower than than with the other ones. Any suggestion on how to improve the assay in this case?
2) for the substrates that I do get a proper M-M-like curve, many times I am not able to obtain good independent replicates. Once the amount of enzyme is not supposed to affect the Km, but only the Vmax, what else could be affecting changes in Km?
3) Also, I've noticed that for some substrates (even if the replicates are consistent) if I consider 7 or 6 different substrates concentrations the Km changes significantly. For example: using substrate from 1 mM to ~ 0.015 mM (7 concentrations) it was obtained a Km of ~ 0.30 mM; while considering just substrate from 0.5 mM to ~ 0.015 mM (6 concentrations) the Km obtained was ~0.17 mM. Someone could explain that or indicate some bibliography?
Thank you in advance!
1. For substrates that are poorly recognized by the enzyme, the Km may be so high that you see little or no curvature in the M-M plot. The solution, if it is practical, is to increase the substrate concentrations further. If this is not practical, then you can at least measure the slope of the straight plot, which is kcat/Km. In the case of a poor substrate, you may have to increase the enzyme concentration to get a sufficient signal.
When going to high substrate concentrations, beware of interference by the substrate with phosphate detection. Make sure to include a no-enzyme blank for each substrate concentration.
2. If you can't get reproducible results, you have to investigate your experimental technique to see where the variation is arising. Is the enzyme or substrate unstable? Is the temperature different between experiments? Is the phosphate detection system giving consistent results with the standard curve? Are you operating in the dynamic range of the detection system? Are you mixing everything thoroughly, but being careful not to damage the enzyme by excessive mixing? Are you sure there are no phosphate residues from cleaning agents on the containers you use to prepare reagents and run reactions?
3. To get an accurate value for Km, you should include substrate concentrations that are at least 5-fold above and below the Km. In other words, you need to see the full shape of the curve to get the most accurate fit.
Thank you Dr. Adam B Shapiro . I'll look carefully at all the points that you mentioned. Hopefully, better results will come!
Dr. Robert Adolf Brinzer , what do you mean by negative controls? some enzyme inhibitor or no-enzyme blanc?
First of all you need to find out the enzyme activity using a fixed saturating concentration of substrate and varying enzyme concentration. You should get a linear curve up to a certain enzyme concentration.
Select the enzyme concentration from the linear portion.
Fix the concentration of enzyme and vary substrate concentration and monitor the constant initial reaction rate.
The lower concentrations of substrate will show a linear response to the reaction rate and then ultimately with increases in substrate concentrations the reaction rate will eventually become constant .
You get a hyperbolic curve.
Line weaver Burk plot will give you the Km .
You will get diff Km value for different substrates if your enzyme is non specific.The best substra is the one with lowest Km value.
If you could send me your measurements, I could try with my software.
See: www.lerenisplezant.be/fitting.htm
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