If an active site mutant knocks out product formation at all excessive concentrations of substrate and at all excessive concentrations enzyme, but substrate binding affinity via anisotropy shows no difference in binding affinity for wildtype versus active site mutant enzyme, is kcat = 0? But if kcat = 0, then by the relationship of Michaelis constant (KM) to kcat then KM = KD.
How do you show to reviewers that the active site mutant is dead? If the wildtype mutant starts producing product in seconds, are you supposed to measure the reaction for the mutant for an hour at zero concentration of substrate and at 100fold excess substrate concentration of the K_M for the wildtype enzyme for the active site mutant, and show the time course?
Are there any publications that show a mutant enzyme is not just slow to produce product but is rather incapable of producing product but can still bind substrate? Examples would be greatly appreciated!
Short answer - I would say yes. If an enzyme can't turn over products on any time scale, its turnover number would be 0.
I agree that if kcat = 0, then effectively your KM is just the ratio of kon and koff, that is, it is equal to the KD. If one was simply measuring binding of the substrate to the enzyme, I can definitely see that replacement of a residue critical to the mechanism, but not the binding and orientation of the substrate, would generate a variant that does not turn over product but still has a similar or identical KD for the substrate. This says nothing about the turnover number (or lack thereof).
How would I show a reviewer that a binding-competent enzyme is catalytically inactive? I think what you have described is going above and beyond. Extending a reaction for 60 min at 100 x KM to prove no activity exists feels a bit extreme. Even if the measured kcat is very tiny but not zero, it would be a moot point as this has absolutely no biological relevance.
If your wild-type enzyme has a kcat > 1 s-1 like you mention, I think it would be reasonable to measure the initial rates of i) the wild-type enzyme, ii) the catalytic mutant, and iii) a matched enzyme-free reaction using the same reaction conditions for all on whatever time scale is appropriate for your enzyme and its relative rate.
With enough replicates, you can use appropriate statistics to compare the grouped samples. Showing that there is no statistical difference in the rate of reaction between an experiment with no enzyme at all versus a catalytic mutant would be a strong argument that no activity exists, i.e. kcat must equal 0.
You will see in this paper we use an LC-MS assay to detect the presence/absence of product in active site replacements as justification for a dead enzyme, in addition to a colorimetric assay where we did the above to justify activity as "n.d." We did not measure affinity to the substrate but the binding site is > 15 angstroms away in this case.
Article Mechanism of Staphylococcus aureus peptidoglycan O-acetyltra...
Hope this helps!
ACA
Adron Ung When kcat is zero, it means that the mutant enzyme is unable to convert substrate into product, even though it can bind to the substrate. This could be due to a loss of catalytic activity or a disruption in the necessary active site residues for the reaction.
To demonstrate to reviewers that the active site mutant is inactive, you could perform the following experiments:
Enzyme activity assay
Product formation assay
Time course analysis: Conduct a time course analysis of the reaction using the mutant enzyme at zero substrate concentration and at a 100-fold excess substrate concentration compared to the wild-type enzyme's KM. If the mutant enzyme fails to produce any product over an extended time period, it further supports its lack of catalytic activity.
By performing these experiments and presenting the data, you can provide convincing evidence to reviewers that the active site mutant is indeed dead and unable to catalyze the reaction, even in the presence of excess substrate.
hope it will clear some-point
Maybe your mutant enzyme lose the catalytic property for the substrate. Did you try another substrate molecule (if it has activity on another substrate)?
If you do not get activity even when you increase the substrate, enzyme concentration or time, then Kcat is zero.
If the substrate binds, then it is possible that the binding is not productive, due to a number of reasons. eg: functional groups may not be in the correct position to facilitate the reaction.
But in your case , since this is a mutant, mutations would have prevented the reaction mechanism from happening, even if the substrate is bound in the proper orientation.
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