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I'm currently working on a project where I have to deal with enzyme inhibition. The purified enzyme shows a good substrate turnover. When I try to inhibit it with different inhibitors described in literature, however, I receive quite awkward results.

I have now tested 3 different inhibitor compounds. One showed good results (it reduced the substrate turnover), while the other two compounds instead led to increased substrate turnover?!

I know it sounds ridiculous, but has someone experienced something like this before? The measurements of the "working" and "not working" compounds take place on the same plate. How can it be that widely described inhibitors lead to such results?

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    $\begingroup$ This is very vague (purity of the enzyme, etc.) You might be messing wit the rate limiting step somehow. But my question is, why aren't you asking someone senior in the lab? Is this a homework question? $\endgroup$
    – anongoodnurse
    Commented Mar 24, 2021 at 16:28
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    $\begingroup$ Adding on to anongoodnurse, you say the rates change but...by how much? What's the variance in your measurements? How many replications? How are replications performed? In what order did you do the experiments and how were the reagents handled during the process? What other conditions are you maintaining constant? $\endgroup$
    – Bryan Krause ♦
    Commented Mar 24, 2021 at 16:30
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    $\begingroup$ What do you mean by a “Signal increase”? This is not a term I have ever encountered in enzymology. Please reword your question using standard enzymological terms. Running your question through an English language spelling checker is also recommended. Modern software and operating systems make this easy. I routinely do checks in French, German and Italian. $\endgroup$
    – David
    Commented Mar 24, 2021 at 19:14
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    $\begingroup$ @David The language was a bit rough, but I found the meaning pretty clear, as well as the starting points for debugging. I have now cleaned it up and hope others will retract their close votes. $\endgroup$
    – jakebeal
    Commented Apr 5, 2021 at 1:47
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    $\begingroup$ @jakebeal I am afraid this question is still completely unclear, and until the poster uses the precise scientific terminology to describe what he observes — together with detail on the so-called "inhibitor compounds" the question cannot be answered. Nor does it deserve to be as the poster has not responded to this basic request to use enzymological terms, leaving one to wonder whether he understands the basics of enzyme measurement and quantitation. The term "substrate turnover" typifies this problem. I spell this out in my next comment. $\endgroup$
    – David
    Commented Apr 5, 2021 at 8:15

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While it's difficult to know just what is going wrong in somebody else's laboratory, apparently paradoxical effects like these are unfortunately common in laboratory work. Assuming that these are indeed well-established inhibitors that really should be working, let's consider why they might not be in your hands.

The mechanisms of molecular interaction are complex enough that there are many cases where a substance that acts as an activator in one condition can act as an inhibitor in another condition. Another way that you could see such an effect is if your purified system is not actually as good as you think and the problem is being partially relieved by one of the inhibitors, e.g., imperfectly purified and addition of the inhibitor neutralizes some of the imperfections.

Not knowing the details of the compounds you are working with, it is not possible to guess which of the possibilities might be at work. I would suggest, however, that you begin by:

  1. Comparing your quantitative turnover rate with the purified enzyme to the predicted rate from the literature to see if your baseline system is actually working correctly.
  2. Make sure your system is also (not) processing substrate at the appropriate low rate when the enzyme is absent.
  3. Checking for potential differences between your method and the method that you are trying to adapt from the literature, which might give clues to the condition differences that may be impacting your results.
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    $\begingroup$ @user338907 Indeed: so many assays go wrong because there isn't sufficient use of process controls or because somebody only checked controls for the qualitative and not quantitative behavior. Your comment reminded me that the OP also didn't mention a negative process control, so I've added that as well, which would detect something like the case you mention. $\endgroup$
    – jakebeal
    Commented Apr 5, 2021 at 10:09
  • $\begingroup$ There's also the factor of error - OP didn't mention the statistics they did on the data they generated, but it is highly likely that the curves are within "no significant difference" of each-other. $\endgroup$
    – bob1
    Commented May 5, 2021 at 20:28

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