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Suppose there is a suicide inhibitor of an enzyme that reacts with the enzyme to form an inactive enzyme and another product. This "Another product", however, is capable of acting as a competitive inhibitor of the enzyme. An example of such an inhibitor is neostigmine, which reacts with acetylcholinesterase to give the carbamylated enzyme and a phenolic product that can also competitively inhibit AChE. If one wanted to assay the reaction rate constant for the formation of the inactive enzyme, how would one determine to what extent an observed decrease in enzyme activity is due to the suicide inhibition versus competitive inhibition by one of the products of the suicide inhibition?

Furthermore, if a studied inhibitor is thought to be able to deactivate multiple molecules of an enzyme (EA-3990 vs. AChE as an example), in a reaction scheme like this:

Initial Inhibitor + Enzyme-->Inactive enzyme+ Generated inhibitor

Generated inhibitor+ Enzyme--> Inactive enzyme+ competitive inhibitor

how could the reaction rate constant for the initial inhibition reaction be reliably assayed?

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  • $\begingroup$ If it is true that the formation of the side product requires suicidal inactivation of the enzyme, seems like all you need to do is measure the rate of increase of the side product, e.g. by LC-MS. Its presence would be directly proportional to enzyme inhibited. $\endgroup$
    – Curt F.
    Commented Jul 25, 2023 at 4:51
  • $\begingroup$ I find the use of the word "assay" a little ambiguous here. An assay is a test, but here you seem to mean "measure". So your question seems to be how to measure the individual rate constants, right? $\endgroup$
    – Buck Thorn ♦
    Commented Jul 25, 2023 at 6:48
  • $\begingroup$ Experimental details would vary depending on the specific system. For example, if the inactivation is a stable covalent adduct, you could quench the reaction and use MS or some other relevant method to determine the fraction of enzyme with adduct at different time points. Or do a dilution/wash to remove the original inhibitor and the competitive derivative and measure activity to determine active/inactive enzyme. $\endgroup$
    – Andrew
    Commented Jul 25, 2023 at 11:56
  • $\begingroup$ @BuckThorn Correct. $\endgroup$
    – user73910
    Commented Jul 25, 2023 at 21:14

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