Are others arguing that you can't make the claim because the inhibition is irreversible due to the double mutation?  Whether or not the the allosteric inhibition is concerted, sequential, or morpheein, is it possible that the doubly mutated enzyme (multi subunit I assume)  does not assemble or fold well enough to even permit  demonstration of possible residual activity?  In other words it may be necessary to produce CD or crystallography data of the double mutant.  Usually these types of allosteric studies are done with ligands as opposed to mutations or with just one of the mutations.  You may already be familiar with aspartate kinase case studies. The argument may be different In the case of allosteric mutations causing receptors to be constitutively "on" in the absence of ligand.

Hi there,

What is actually the location of the mutations? Are they both within the active site of the enzyme or outside and are they close/distant from each other. This is the issue to address in order to check if these mutations affect totally different steps of the reaction mechanism or if they affect the same step... However additivity of mutation effect doesn't prove anything about the reaction mechanism.

Hi Gary

I agree with Dominique Liger for his comment. My question: what is it the quaternary structure of your enzyme? monomer, dimer,....?

Thanks for your feedback

It is a dimeric enzyme, the structure, stability and conformational dynamics of the enzyme have not altered due to the mutations. Both mutations are at the active site.

Both mutations singularly reduce the activity by approx 90%. together the mutations inactivate the enzyme. 

I will see if I can find the paper I am thinking of, but the author argued that since the effect of two mutations was excatly additive that their steps must be concerted. 

Hi again,

It can't be concluded that the effects of the mutations are strictly additive since, as each mutation inhibits 90% of the activity, the additivity would result in 180% inhibition, but you can't observe more than 100% inhibition...

Imagine the 2 mutations being strictly independent (affecting 2 different steps in the reaction mechanism), the double mutation should result in 99% inhibition (the second mutation affecting 90% of the 10% residual activity resulting from the first mutation)  which I guess cannot be distinguished from a total inhibition.

For proving the additivity of the effect of mutations, addition of individual mutations should be less than 100% inhibition (and ideally not more than 50% each) in order to be able to make the difference between additivity and combination of mutations. Let's say each mutation affect 50% of the activity, the double mutant should exhibit 0% activity if additivity and 25% activity if mutations are strictly independent.

In your case, you should try to construct mutants with less drastic effect on enzyme activity. 

If you're not doing allosteric studies then are you moving to substrate binding constants and kinetics for these three mutant forms?  Probably won't be as simple as being able to change an inhibitor's concentrations but maybe a combination of a single mutation with an active site inhibitor?

Hi all

The posed question and all the precedent comments are of interest. But, data regarding the nature of the mutation and the kinetic of the native enzyme (E) are lacking. Does the E reaction obey to allosteric or Michaelis-classical kinetic? In any case, Gary can check experimentally the effect of the two mutations: complete inhibition or some additive one?

-Assume that X min is normally applied to measure the native E.

- In the case that the E is inhibited additively, 10 X min must be applied at least to detect residual activity.

- In the case the E is allosteric, some activity (e.g. 10%) may be remaining which could be measured with prolonged reaction time. Besides, inhibition could be complete.

- Deep kinetic studies (comprising crystallography) must be performed in order to establish a useful model.

I have to agree with Dominique. How can the author of the paper claim the two mutations are "exactly additive" if each mutation confers 90% inhibition? If both mutations are in the active site, one could infer significant activity loss by each mutation, but in this case, I doubt one can claim additivity when 90 + 90 = 180% activity loss. I assume substrate concentrations are already saturated?

As for activity loss being a "concerted mechanism", these mutations may give inactive conformations in the active site (coordination ligands, or merely tertiary structure disorganization) that are not directly tied to the 'enzymatic mechanism'. But possibly the term 'concerted mechanism' includes any conformational disorder resulting in activity loss. A matter of terminology.

Additivity of inhibition does not mean 90% per mutation 1 + 90% per mutation 2  =180%. It is obvious that this consideration is not logic (commented by Dominique Liger). It is possible to admit that the two mutations cause a 100% activity loss or less than 100% (e.g. around 95%). In this, each mutation causes inhibition of 50 % or less (e.g. around 47.5%). Data regarding the chemistry of the active site, the nature of the mutation (which amino acid(s) changed) and the enzyme kinetic type are lacking.

Q: the mutated enzyme activity was it assayed with a sufficiently prolonged reaction time?

Rather than using percent reduction in activity as a measure of the effect of mutations, it would be preferable to measure the effect on substrate Km, kcat, and kcat/Km. If there is cooperativity in the mechanism, one could also look at the effect of the mutations on the Hill coefficient. There might be other effects as well, such as on the pH dependence and the activation constant for any metal ions that might be cosubstrates. These are the sorts of details that can be used as clues in the investigation of the chemical mechanism.

I don't know what it would mean to say that the effects of 2 mutations on these parameters were additive, sub-additive (antagonistic), or super-additive (synergistic), and I can't figure out how such information would reveal whether a mechanism was concerted (2 bond breaking or forming steps occurring simultaneously).

Here is an example of a paper that uses individual site-directed mutants to investigate the chemical mechanism of an enzyme and support a concerted mechanism.

https://bib-pubdb1.desy.de/record/84445/files/winkler_et_al_final.pdf

This is the reference: Nature Chemical Biology 4, 739 - 741 (2008)