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My laboratory team has been used succinate dehydrogenase (1.3.5.1) from Gallus gallus domesticus breast (the most meated part of the chicken, where you will obtain nuggets) to determine $K_{m}$ and $V_{max}$ under 3 linearization methods.

But, in BRENDA page, I found various values for many organisms, like Bos taurus, and they select other tissues like liver tissue.

Why the difference?

PD. Any reference about where to find this values for Gallus gallus mesticus will be very appreciated.

EDIT: The reaction consists on dehydrogenating succinate to fumarate, on the tricarborxilic acid cycle. Include the creation of a trans-double bond.

Describing my laboratory practice, we macerate the meat with glass sand, and divide this mixture on 2 kinds: one with methylene blue and trichloroacetic acid (TCA) and one without TCA. Now I know that TCA is a protein precipitant, and methylene blue is an indicator; the less blue, the less succinate. So, with absorbance tests, we can elucidate [Succinate] vs time plot, and approximate the ratio $V_{max}/K_m$ from the michaelian equation for substrate concentration decay: $$S(t)=S_0 e^{-\frac{V_{max}}{K_m}t}$$ Also, i think it will be possible modeling instantaneous velocity with its derivate and make the linealization plots, but i'm not very sure of this.

EDIT2: Thanks for the help with my practice report (I really appreciate it), but I still wanna know if it's possible to have different $K_m$ values on the same enzyme, only changing the tissue for reaction.

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    $\begingroup$ Can you please edit your question to add an example of such enzymes from BRENDA? $\endgroup$ – WYSIWYG Oct 11 at 7:45
  • $\begingroup$ Also note that succinate dehydrogenase is a part of a complex and catalyzes many coupled reactions. A simple MM kinetic model won't apply in this case, I believe. Which reaction are you interested in? $\endgroup$ – WYSIWYG Oct 11 at 7:55
  • $\begingroup$ As far as I know Brenda lists experimentally determined values, if they come from different studies/sources the differences are much more likely to be due to that than due to different tissues $\endgroup$ – Nicolai Oct 11 at 13:45
  • $\begingroup$ I need experimentally values reported, so data in BRENDA is usefull. But I duno why it will be different values on different tissues. This is not possible, under the same conditions? $\endgroup$ – Adonis Cedeño Oct 11 at 18:16
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    $\begingroup$ If the enzyme is not pure (are you dealing with tissue extracts?) then it is not surprising that Vmax for the enzyme should differ when the enz is extracted from different tissues as Vmax will depend on protein concentration, and differences in the simplest case may merely reflect different levels of SDH in different tissues. If a liver contains 10 times more SDH than a brain extract, then the Vmax of the liver extract will be 10x that of the brain extract. The same applies to Vmax/Km, but Km of course is independent of enzyme concentration (even in a crude extract) $\endgroup$ – user1136 Oct 11 at 20:39
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You would not expect the same enzyme from different tissues to have different Km values if the enzymes are truly identical.

In addition, you would not expect the Km value to change during purification: the Michaelis constant for the purified enzyme should be the same as that determined with a crude sample. If this is not true, it might mean that the enzyme has become modified during purification, perhaps by proteolysis (not something you want your examiner to point out or suggest!).

One advantage of the Michaelis constant is that it is independent of enzyme concentration (and so does not depend on the degree of purity).

Having said that, I think it would not be considered too unusual for enzymes isolated from different tissues to have different Km values.

Possible reasons are:

  • The assay conditions are not really the same. pH, ionic strength, temperature and buffer composition may all affect Km. The Michaelis constant determined at pH 7 in 100mM phosphate at 37oC may be very different from that determined in 50mM Tris pH 7 and 25oC
  • Post-translation modification may be important. The enzyme from one tissue may be phosphorylated, for example. One laboratory may have isolated the modified from, whereas another laboratory may routinely include phosphatase inhibitors.
  • Proteases present in the crude tissue preparation may have modified the enzyme.

In the case of succinate dehydrogenase, there are two other important considerations.

  • Succinate dehydrogenase is a membrane-bound enzyme (containing a covalently bound flavin) and the method of extraction will be all-important, and a key area to examine is whether extraction conditions may account for the difference in Km.
  • Finally, and this one is important I think. SDH is a two-substrate enzyme and the Km for substrate A will depend on the concentration of substrate B ! To determine the 'true' Michaelis constant for a two-substrate enzyme (assuming no substrate inhibition), we must extrapolate to 'infinite' concentrations of the co-substrate. Otherwise we are dealing with apparent Km values.

I'll bet the last point is important. Is the problem that at least some of the reported Kms are apparent values, ie the concentration of co-substrate is different under the different experimental conditions?

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