I am working on an enzyme, which Km I have calculated after its rate saturation. I have solved the crystal structure of this enzyme and wanted to perform a mutation in the active site based on its structural moeity of its coenzyme binding site. The residue is important for coenzyme recognition. After mutation when started its saturation, it show a linear increase in change of absorption. Even if I reached to millimolar range it, the Vmax is not reached. I have taken very less concentration of enzyme. Of course this reflect very high Km value, which cannot be measured by UV/Visible spectrum, because the highest concentration of substrate reached till 0.98 Abs. Beyond that how can I go further. Please suggest how I can I proceed to calculate it's Km value.
Hi there,
If you data show that hyperbolic relation between rate and substrate concentration can be established then fitting the data with hyperbolic equation will give you both Km and Vm using non linear regression. There are non commercial website where you can run this for free:
https://mycurvefit.com/
http://faculty.gvsu.edu/carlsont/232lab/nonlin2.html
Sorry -- unable to help you -- however:
Begin by clicking on to my video presentation:
https://youtu.be/-MEMM_-kUWg
After this go to my website:Â Â Â Â Â http://henrymsobell.com/
clicking on to Book 1, Book 2, along with my JSFB (2016) paper.
Many thanks for your question.
Henry M. Sobell
@ Dear Liger
I have already calculated the Km of the wild type, as it fitted quite well in the hyperbolic (non linear regression) but my problem is the reviewer has asked us to calculate the Km of the mutants as well, but one of the mutant is not reaching to the saturation and the relative activity is less than the wild type, that obviously reflect very high Km for this. When I go increasing the concentration of the substrate it is linearly increasing. (no matter higher substrate concentration is increased very high compared to [E]. At certain point the absorbance reaches to 0.98 so I cannot go beyond that to measure the Abs. of the reaction. So is there anything that can help me out there. Thanks in advance looking for your kind suggestions.
The standard method of measuring Km using the Michaelis plot, or a linearization of it, does not work if there is no sign of saturation due to a very high Km. You can try to increase the dynamic range of the assay by shortening the path length. For example, if you are using a 1-cm cuvette and the absorbance is too high, switch to a 2 mm cuvette, allowing you to increase the concentration 5-fold. Also, try to find a spectrophotometer with a larger dynamic range. Some of them are linear up to an absorbance of 3, rather than 1. Put both methods together and you can increase the substrate concentration 15-fold.
Another approach is progress curve analysis. In this method, instead of making initial rate measurements, you follow the reaction out longer until it slows down due to substrate depletion. You then numerically fit this progress curve to the integrated form of the Michaelis-Menten equation. Ideally, you should do this at several substrate concentrations. The Km and Vmax can thus be obtained. Care must be taken to understand whether there is also product inhibition.
Dear Adam
Thank you so much for enlightening me about the progressive curve analysis. But if I want perform this, how should I design the experiment for this and how reliable it is (as whether it is acceptable in enzymology because mostly people rely on rate measurement. I will be grateful to you if you suggest the protocol of doing this and I can perform this experiment as soon as possible. Thanking you ,
Prem
Hi again,
do you absolutely need a numeric value for this Km? If there is no sign of slope weakening whatsoever in your Vo=f([S]o) graphics then you may estimate than your largest concentration of substrate considered is still far less than the Km (at least 10 times less)... So you may compare this low estimate of Km with the one obtained for the wt enzyme. If the finality is to show that the mutant is physiologically irrelevant because its Km is far more than the physiological concentration of substrate and that the velocity of the reaction catalyzed by the mutant is very low compared to the wt at this physiological concentration then the estimated Km should be enough
Hi Liger,
Thank you once again for your kind reply. It will helpful for my learning. But one thing still has to be clear to me that can this assumption of 10 times lower than Km value will be acceptable bcz we cannot extrapolate the maximum value from this experiment.
There are a number of published papers on the subject of progress curve analysis. Here are a few:
Article Analysis of enzyme progress curves by nonlinear regression
https://link.springer.com/content/pdf/10.2478%2Fs11535-008-0035-4.pdf
Preferably, you should have a continuous measurement of product formation or substrate consumption, rather than individual time points. You will have to experiment with the enzyme concentration to get a set of progress curves that show curvature during a reasonable time frame of the measurement. You should test the potency of the product(s) for inhibition of the reaction, because you need to know if product inhibition is occurring. You should make sure the enzyme is stable during the whole reaction time.
You will need a nonlinear regression program to do the data fitting to the integrated M-M equation. Alternatively, you can use a numerical integration package for enzyme reactions, such as DynaFit or Kintek Global Kinetic Explorer.
Hi again,
To see if my assumption is acceptable you should tell me what the wt Km is and what is the highest concentration of substrate tested with the mutant? Also if possible the rate of the reaction measured for both enzyme with physiological substrate concentration.
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