You can do lots of experiments to see if there is difference between native and mutant enzymes. As already suggested you can do pre-steady state kinetics, pH optimum, temperature optimum, irreversible thermal inactivation studies, secondar structure melting temperature using Far-UV CD, Tryptophan intrinsic fluorescence spectral analysis and melting temperature, differential scanning calorimetry and melting temp. I am not aware of your enzyme or substrate but may be it does not show difference with one substrate but may show difference with another substrate. You can try Natural substrates using isothermal calorimetry (iTC) if its difficult to assay by conventional colorimetric assays. May be your mutant is inhibited or activated differently etc.

A near 10-fold change in Km in my opinion is already a significant change. Of course these are things you should assay several times and in different temperatures etc. to gain statistical relevance. A doubled Km to my understanding is already regarded as an indication of retarded binding. I assume your kcat does not change? When working with enzymes in mutational studies I think it's always good to have at least one mutation that abolishes activity, for the sake of controls. This at least helps in verifying the essential catalytic amino acid(s) and sometimes even the mechanism of action. Anyway, the kinetic parameters that are usually compared between mutants are kcat, Km and their ratio. I find kcat/Km a very informative one in case you want to check how the enzyme efficiency changes.

EDIT: Oh and should I also mention that whilst working with mutants, at least what I have done in the past, it is good to check the structural integrity of your proteins by methods like CD, SEC and SAXS - just to rule out possibilities that your mutation causes a significant change in conformation and thus deletes activity. It is also good to verify that you have the correct mutation by mass spectrometry, for example. MALDI-TOF combined with trypsination is very useful as it gives quite specific information at the amino acid sequence level.

Regarding the kinetic changes I agree that you have to expect at least a 10-fold change (up to 10^3/10^4 changes are commonly observed in enzymes that maintain their activity). On the other hand, even few °C of difference in Tm could be interesting.

If you are dealing with a series of variants and kinetic parameters, stability, oligomerization state and CD spectrum are similar to the WT, than I'll just assume that you did not touch any critical residue and I'll move over.

To check if you are working with the correct variants just check the nucleotide sequence. It is more simpler and economic than checking MW of the protein by MS.

Gianluca

P.S. sometime is worth to check if substrate specificity changed.

I agree with former comments, but the decision about when difference is significative is difficult. 10-fold is already a good limit, but 5-times would be also significative to me. It depends on the enzyme and the implications of this change to the physiological situation. Sometimes, to chanhe 5-times something is dramatic, and sometimes is not important.

On the other hand, you talked about Km, but what about Vm (Kcat) and the efficiency ratio kcat/Km? This could be more valuable and informative.

Obviously, mutations at the active site are more important for this parameters, but if your enzyme is allosteric, and it cotains other sites for regulations, this should also be inestigated in the presence absence of effectors .... if ther are known effectors.

Cheers and good luck,

Do you have a method for active site quantification for your enzyme? This is necessary for correct kcat measurement, absolutely essential.

What do you mean with "active site quantifitation"? For kcat determination, she will need Vm (reaction rate at saturating concentrations of the substrate) and the molecular concentration of the enzyme.

Dear Anne Makena ,I agree with above comments given by many experts. In my opinion even in some mutation 5-10 fold increase in Km are also observed but not necessarily that the target residue is strictly essential for the catalytic activity. As said above,Km/Kcat would be an important criteria to check, if the various mutation brought does really affects Km/Kcat;catalytic efficiency of a enzyme.Certainly, it also depends on the kind of residue you substituting with. Substitution with similar class residue mostly not change the Km or Kcat to a great extent. In contrast, resdiue with opposite charge/property would either led to loss of activity or show activity sometime at very higher substrate/cofactor concentration(decreased Km,Ks,Kcat ). Therefore, you can always try mutating some residue by two or three various class of residue and then reach out to some conclusion about the role of certain residue. If you have done so, Yes, preliminarily you can rule out /assume that this residue may not be so crucial or strictly essential for the catalytic function of your enzyme. Further, i wonder, mutant enzyme at single point mutation with slight increase in Km would reflect any major difference in CD spectra between wild/mutant enzymes. But yea, you should check through CD studies,if there is any slight conformational changes up on mutation in your enzyme. In addition, would like to share my personal eperience in dealing with such mutants. Recently i have studied one important residue in one enzyme by substituting it with similar or different residues. I observed that, if the residue are highly conserved and strictly essential,it would not reflect any activity irrespective of any type of mutations you bring. However, it may reflect certain degree of activity at the expense of increased substrate/cofactor concn;resulting into decreased Km,Km/Kcat. Thus, in such cases,based on the such altered kinetic paramater you may get some prior indication/clue that the target residue might have active catalytic role/function. Further structural characterization (CD,molecular dynamic simulation) study would be more helpful to support your results/observations.

Please feel free to correct me, if somwhere i went wrong.

Good Luck in your research endevours.

Regards,

Irshad Baig

PhD Student.

Many thanks for all your very informative answers.

Karim Engelmark, I dont quite understand what you mean by active site quantification. Accrding to the crystal structure, the enzyme has one catalytic site. The mutations am looking at are found in nature but are not located in the active site of the enzyme. It is possible that they have no effect on the catalytic activity and maybe they are just stabilising mutations.Has anyone got some reference where peripheral mutations (found away from the active site) have affected the catalytic profile of an enzyme?

Dear Anne, perhaps you could have a look at this article, which outlines the role of a conserved off-active site arginine residue required for the enzyme to function. http://www.ncbi.nlm.nih.gov/pubmed/14659757

You might also check if any changes have occurred in the pH dependence of activity, in particular if the mutation implies substitution of aminoacids able to dissociate.

As people noted above, I think the first thing you want to do is determine whether the mutations have effected the structure of your enzyme. CD is a fairly quick and easy way to do this. Then you want to figure out if the changes in Km you are observing are actually significant. As someone noted above, you want to repeat the assay multiple times, preferably using enzyme from more than one expression/purification. Then do a statistical analysis to test for significance (e.g. two-tail t-test). Without some kind of analysis, or at the very least error estimates, you can't really say whether changes in Km from 2 to 12 are significant.

Francisco, of course she needs to measure the Vmax. But, if she uses total protein (without measuring the amount of active enzyme) she will obtain the specific activity, which is not a suitable number to compare the variants by. The true kcat is based on active enzyme, so if she makes a bunch of enzyme variants (mutants) the active enzyme fraction can by no means be expected to be the same; this is also true for the same enzyme in different cultivation batches.

I don't know if this is helpful or not, but sequence differences distal to the active site can affect enzyme activity at low temperatures.

http://www.jbc.org/content/286/44/38348.full.pdf+html

Thank you very much Irshad. I've checked the mutants CD spectra and they seem pretty similar. I checked the masses using MALDI and they correlate well with the expected masses. I'll compare the catalytic efficiency (Kcat/km) and probably do some simulations...

Dear Anne;

Usually steady state parameters are just starting point but traditionally used to characterize enzymes. If you dont see any significant changes in catalytic parameters of your mutants, you have to try pre-steady state kinetics. These experiments are not trivial and not many labs do it. Basically, in presteady state, your enzyme and substrate concentration are in similar range and you go in the msec/sec time region to check for enzyme activity. It will also tell you how much enzyme in your solution is active . Instead of getting Michaelis Menten parameters you will get real activity numbers and will be able to surely characterize the mutants. These experiments are well characterized for protein kinases and I have listed two publications.The second paper is where the mutant had similar kcat to the wildtype enzyme but pre-steady state kinetics showed it was deficient in phospho-transfer capacity. You use an instrument called Rapid Quench to do pre-steady state experiments and also radiolablelled substrate. http://www.ncbi.nlm.nih.gov/pubmed/8639687; http://www.ncbi.nlm.nih.gov/pubmed/22334660

You can do lots of experiments to see if there is difference between native and mutant enzymes. As already suggested you can do pre-steady state kinetics, pH optimum, temperature optimum, irreversible thermal inactivation studies, secondar structure melting temperature using Far-UV CD, Tryptophan intrinsic fluorescence spectral analysis and melting temperature, differential scanning calorimetry and melting temp. I am not aware of your enzyme or substrate but may be it does not show difference with one substrate but may show difference with another substrate. You can try Natural substrates using isothermal calorimetry (iTC) if its difficult to assay by conventional colorimetric assays. May be your mutant is inhibited or activated differently etc.

Did you express the proteins using a tag, and did you remove the tag before testing the activity of the enzymes? I have seen a His-tag stabilize a mutant protein such that is was as active/stable as the wt enzyme, however, after removal of the His-tag from both the wt and mutant proteins the mutant protein became very unstable as predicted based on modeling studies.

What Karin was discussing was for titration of your activity with a tight binding inhibitor. Do you for example just use total protein to determine kcat/ Km or do you measure the amount of active enzyme with a tight binding inhibitor?. If you truly measure kcat/Km, then you will do the latter. The kcat/Km could vary significantly if for examples most of your protein is dead and inactive.