Hi,
In an enzyme kinetic experiment, my result showed a consistent decrease in Vmax by increasing magnesium but Km fluctuated. Till 40mM Magnesium, Km decreases, but it was increases at 60mM magnesium and again decreases by further increasing magnesium to 100mM.
I think both these parameters are independent and Km is a complex parameter that depends on various factors and internal enzymatic properties and therefore it can vary.
I will be grateful for opinions and suggestions.
First, ask yourself whether these Km fluctuations are reproducible and of a significant size, or whether they might just be within the variation of the measurement.
There are multiple reasons why changing the Mg2+ concentration could affect the kinetic parameters. If Mg2+ is cosubstrate (i.e. the actual substrate is the Mg2+-substrate complex), it will obviously have an effect on the Km and Vmax. For ATPases, as an example, it is not unusual for there to be an optimal Mg2+ concentration for Vmax, which is the amount needed to saturate Mg2+ binding.
Exceeding this concentration can reduce the activity of the enzyme. This could be because of the chaotropic effect of the high ionic strength of Mg2+, or because the pH is shifted (MgCl2 is acidic, for example), or Mg2+ competes with substrate. Mg2+ could also have an allosteric effect by binding elsewhere on the enzyme than the active site. Such effects could change either Km or Vmax or both.
The concentrations of Mg2+ you are using are unphysiologically high, so it would not be surprising to find that Vmax and/or Km were different that they are at lower concentrations (5-20 mM).
Adam B Shapiro and Sourav Roy
My Vmax consistently decrease by increasing Mg from 10mM to 10mM and this decrease is significant. But Km value not decrease or increase in a consistent manner. The results are reproducible.
Km Vmax
8.19±0.598 (10mM Mg) 14.97±1.002
7.66±1.15 (20mM Mg) 12.19±0.48
10.05±0.215 (40mM Mg) 12.19±0.239
9.34±1.40 (60mM Mg) 10.60±0.37
4.60±0.37 (80mM Mg) 6.82±0.33
8.19±0.598 (10mM Mg) 14.97±1.002
Can it happen?
My interpretation is that these differences are small enough to be within the range of experimental variation, with the possible exception of the 80 mM one, which is an outlier. Are you showing standard deviations or standard errors. Are these values for independent experiments?
Adam B Shapiro
I am showing standard error and these values are of 3 independent experiments.
Sourav Roy
Thank you for the suggestion. I have used 10mM for all enzyme assays. But I am curious about how multiple binding for Mg can change the Km which is specifically for the substrate (GTP).
Interesting!
if the enzyme has multiple binding site for Mg2+, could there possibly a cooperative binding taking place? but the Km values are unfortunately inconsistent to draw that conclusion.
It would be more informative to show the standard deviations of the averages of independent measurements of the kinetic constants if they were measured multiple times. This will give a better idea of the experimental variation.
Some substrates like nucleotides form complexes with Mg. In Mdr1, for example, substrate is the Mg-ATP complex, free ATP is inhibitory. In addition, high salt concentrations reduce the available [water], and hence mobility of the enzyme molecules and hence turnover number. As result, the kinetics of Mg can be complex.
Thank you Engelbert Buxbaum
I also think the same. Magensium and NTP conjugate is a complex structure and therefore it showed such type of result.
Km and Vmax are not entirely independent of one another. Both are functions of the forward rate constant k2. Moreover, modifying the enzyme active site is likely to affect both of these parameters, often in the opposite direction to one another.
Bernard R. Glick Engelbert Buxbaum Adeyemi Oluwadare AyodejiSourav Roy
Adam B ShapiroI really appreciate all answers. To clarify the doubt over this, can anyone of you please provide me with scientific literature that can prove the fluctuation of kinetic parameters with different concentrations of magnesium ion.
Article An Open and Shut Case: The Interaction of Magnesium with MST Enzymes
One is the M-M equation based on certain assumptions, and the other is the method of determining the parameters. In the equation, each parameter is strictly defined, while by the method of determination they are numerically correlated. Of course, the value of the parameters depends on the substrate, enzyme, process conditions (T and pH) and the different substances added.
Hi. I hope the following report might help very much:
https://ocw.mit.edu/courses/biology/7-014-introductory-biology-spring-2005/readings/l5_bioche_deltag.pdf
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