We have been working with an inhibitor that clearly and consistently reduces an enzymes product by about 25-30%. However in trying to elucidate the mechanism using Michaelis-Menten and Lineweaver-Burk, we see no significant difference between Km and Vmax for untreated vs treated (inhibitor added) samples?
If the inhibitor irreversibly damaged the enzyme, would this explain these results?
25-30% is a pretty small effect. It might be difficult to observe a change in kinetic parameters if there is much scatter in the data points.
You should see an inhibitor concentration dependence on the % inhibition. If 25-30% inhibition is the highest you can get, it may be that the inhibitor is not soluble enough in the buffer to get the concentration in solution high enough to exceed that level of inhibition.
Partial inhibition is a real possibility for an allosteric inhibitor. On the other hand, apparent partial inhibition could also be due to a non-specific (i.e. non-stoichiometric) mode of inhibition, or to interference by a non-inhibitory compound with detecting product formation, or to an interaction between the inhibitor and the substrate rather than the enzyme.
Thanks Adam. We did increase the inhibitor concentration and were able to obtain 98-99% inhibition. However at that concentration of inhibitor the pH changed pretty significantly too and we know that part of the inhibition was due to pH alone. We do think there may be some non-specific reactions occuring with enzyme and inhibitor and possibly substrate and inhibitor. Our next step right now is to test if the inhibition reaction is irreversible. Any suggestions on how to test other than Michaelis-Menten and Lineweaver-Burk?
Calculate the Hill slope of the IC50 curve. For a specific 1:1 binding stoichiometry, the Hill slope is usually close to 1. A significantly higher value (e.g. 2) usually indicates a non-specific mode of inhibition. (There may be exceptions for complex enzymes with cooperative binding sites.)
The following test has proved very useful in my experience to identify non-specific inhibition. This idea was based on the work from Brian Shoichet's lab on aggregator-type non-specific inhibitors, but I think it is also good for chaotropic compounds.
This method combines a test for time-dependent inhibition and a test for enzyme concentration-dependent inhibition, both of which are observed for weak, non-specific inhibitors, and were observed for aggregators.
Measure the IC50 of the compound under 2 sets of conditions: a long reaction, such as 30-60 minutes at a low enzyme concentration, versus a short (2 or 3 minutes) reaction at a high enzyme concentration. In both cases, it is essential that initial rate conditions apply and that the enzyme concentration is well below the substrate concentration. The substrate(s) concentration(s) should be the the same for both conditions.
The IC50s should be about the same under both sets of conditions if it is a specific inhibitor. If the IC50 increases significantly under the high-enzyme/short reaction condition, there is a strong likelihood it is a non-specific inhibitor.
You should also include 0.01% Triton X-100 detergent in the assay buffer. This helps to prevent inhibition by the aggregator-type of compound.
This test is intended for relatively weak inhibitors, such as those with IC50s in the µM range, and is only relevant for inhibitors that ought to be in rapid equilibrium with the enzyme. It obviously does not apply for mechanism-based or covalent inhibitors. Also, if the enzyme is known to show specific, time-dependent inhibition due to a conformational change, you should not use the test.
If the inhibitor is changing the pH of the reaction, consider neutralizing the inhibitor and/or increasing the concentration of the buffer to resist the pH change better.
To check for interference with detection of the product, you can prepare a separate set of mock reactions in which the enzyme is omitted but the product (or a surrogate for it) is present at the same concentration at which it is usually formed. You will then be able easily to see whether the signal is decreased by the compound. You can also use this information to correct for the interference.
It is also useful to have an orthogonal assay for the reaction, meaning one in which product formation is measured in a different way, especially if it involves a separation step so that the compound can't interfere with the readout.
I see the possible causes which I explained as follows
Thank you Adam and Mahesh. We have explored some of these solutions but will definitely consider some others that you both have proposed. Hopefully you might be available for more feed back once we have progressed a little further. We really appreciate your time in responding. We are a very small lab with undergrad research students in an isolated area so finding help with this type of question is challenging.
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