Dear all,
I got results after performing an enzyme kinetics with 4 different substrate the kcat of B and D is respectively two times lower than A and C. However, I am seeing the higher Km of A i.e 174 nM as compared to B i.e 54 nM. Can anyone please suggest me how to rationalize this result ? The Kd value however, is always doubled for A and C when compared to B and D respectively. Below are the data attached for your reference. How could it be explained the research paper ?
Thank you
The different kinetic constants for the different substrates reflect the chemical differences between the substrates, including the strengths and types of their interactions with the enzyme active site and differences in the activation energy of the reaction. The differences in the kinetic constants between the substrates in this case are actually quite minor, and could easily be within the range expected for experimental variation.
Adam B Shapiro Thank you so much Dr. Shapiro for your invaluable input here. Initially I thought the same that the difference is not significant but the experimental replicates have shown that Enzyme degrades the substrate A faster than that of B. Likewise C was faster than D. Then I plotted each kcat values as bar graph and calculated the p-values (for kcat) of the experimental replicates and the p values values were in the range of 0.0007 to 0.0234. So the Kcat is two fold higher in the above cases. Then we performed molecular dynamics simulation with those substrates and we found stark differences in the binding mode of two comparative substrates into consideration. Which suggest why one substrate is favorable over the other. The image I have attached for your reference which have been reproducible so far.
Hi there,
By definition, Km should always be at least equal to or above Kd... Which is not the case for substrates b, c and d...
Dominique Liger Dr. Liger, Thank you so much for your valuable input. Yeah ! That's right and I had the same opinion, but then I dug more into the details and I found this book chapter by Jon R. Lorsch (Methods in Enzymology ; Chapter 14) (attached). It is not necessarily to have to correlate Km and Kd because of several factors. What I found here interesting and that makes total sense in my case. Because in my case, the cofactor of the enzyme is Mg2+ which is required to accomplish the catalysis. However, to determine the Kd I used method of fluorescence polarization anisotropy, where I must halt the reaction and measure the binding strength of the DNA substrate with this exonuclease enzyme. Therefore, I have used Ca2+ ion instead of Mg2+ (same group metal ions, but atomic size is different) to give the similar condition but not the identical. Which may have the effect and differed values of Kd and Km. But yes ! I agree with you it should be the case in general.
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