1
$\begingroup$

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.

$\endgroup$
8
  • 1
    $\begingroup$ Can you please edit your question to add an example of such enzymes from BRENDA? $\endgroup$
    – WYSIWYG
    Oct 11, 2019 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, 2019 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, 2019 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? $\endgroup$ Oct 11, 2019 at 18:16
  • $\begingroup$ user1136 thank, I didn't know about the protein precipitation effect of TCA. That changes everything, since the Rx have a non-competitive inhibition. $\endgroup$ Oct 12, 2019 at 3:00

1 Answer 1

5
$\begingroup$

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?

$\endgroup$
6
  • $\begingroup$ One advantage of the Michaelis constant is that it is independent of enzyme concentration (and so does not depend on the degree of purity). This is strictly true in the limit of dilute enzyme concentrations and saturating substrate concentrations, $[S]_{tot} >>[E]_{tot}$ which is usually the case but not necessarily feasible at all stages of purification. If Km is determined in subsaturating conditions (or concentrations that cause protein aggregation) it will apparently change over the purification. $\endgroup$
    – Ryan
    May 30, 2023 at 16:47
  • $\begingroup$ "... which is usually the case but not necessarily feasible at all stages of purification" ?? In my experience, [S]o will always exceed [E]o at all stages of purification This is one of the simplifying assumptions of the steady-state approximation, under which Km is usually defined. Can you give an example where this is not feasible during purification. (ii) "If Km is determined in subsaturating conditions" Subsaturating concs of what? Of [S]o ? This has to be the case if a rate is determined at sub-Km concentrations. Of [E]o?? But the usual assumption is that Eo << So (always) $\endgroup$
    – user338907
    May 31, 2023 at 16:23
  • $\begingroup$ Can you give an example where this is not feasible during purification. If the substrate has low solubilities (for example, gualylates, methyxanthines) then at high enzyme activities the maximum feasible substrate concentration can be subsaturating. If the enzyme has a high specific activity, for example superoxide dismutase, then obtaining saturating substrate concentrations can also be difficult. There are some obvious technical solutions to these problems but, if they aren't implemented correctly, the apparent Km will change over the purification. $\endgroup$
    – Ryan
    Jun 4, 2023 at 0:52
  • $\begingroup$ If the substrate has a low solubility it may be impossible to determine the Michaelis constant at all stages of purification if the highest conc of substrate that can be obtained in the assay is way below Km. But this does not mean that the Km has changed during purification. As long as assay conditions differ only in the degree of purity of the enzyme, and as long as the steady-state approximation applies, one would not expect the Michaelis constant to change during purification. The purified enzyme will be typically diluted to ensure steady-state conditions, for example $\endgroup$
    – user338907
    Jun 4, 2023 at 6:09
  • $\begingroup$ i didn't say Km actually changed I said it can apparently change over a purification. $\endgroup$
    – Ryan
    Jun 5, 2023 at 0:47

You must log in to answer this question.

Not the answer you're looking for? Browse other questions tagged .