To evlaute the effects of mutations in the active site, you need to measure two kinetic parameters: Kcat and Km and then compare the catalytic efficiency (Kcat/Km) of the wild-type and mutant enzymes.

Dear Ho Sup Yoon, thanks for your kind response to my query. Yes i did measure this two important kinetic parameter of catalytic effeciency.. I found the mutant showed reduced catalytic potential compared to wild enzyme..

In addition to kcat/Km value, the structural reason to keep the activity is that, although the affinity of the enzyme for its ligands is lower than that for the wild-type enzyme, the enzyme still binds the ligands with higher Km and Kc. Therefore, increasing the concentrations of ligands will increase the concentration of the active ternary complex.

As Ho has pointed out, you should vary the substrate concentration and measure the corresponding activity of wild-type and mutants. This allows you to determine Km and kcat. An active site mutation should result in an increased Km and/or a decreased kcat, but not necessarily in a complete loss of activity (although this is possible). If e.g. a residue is involved in substrate binding, but not in the catalytic event itself, this will increase Km without affecting kcat. There are also examples where adjacent residues can substitute for a mutated catalytic residues and hence restore some degree of activity.

For the interpretation, you first do have to consider the charge: Glu to Asn or Gln means you are going from a negative to positive charge, hence you destabilize the H-bonding probably required for stabilizing your substrate or an intermediate during catalysis. As a resut, you do impair at least KM. If the charge is also required for catalysis yuou will also impair the kcat. Glu to Ala results in the same as you create a "hole" in your active site and impair catalysis as the substrate might not be able to bind. With Glu to Asp you do change the length without affecting the charge and you thus might have a better kcat/KM. As Asp is shorter, it might impair a bit your KM. As you do mutate an important residue, and a charged one, you might want to check that your enzyme is as stable as the wt, e.g. denaturating curves obtained by fluorescence or CD to have a Tm. The pKa(s) of obatined by plotting the activity as a function of the pH should also be affected.

What I do not understand though is that variation in your activity test. Anyway. If you do have a cofactor, you should also start from the "apo" form and, under substrate saturating conditions, determine with your activity test a kact (activation constant) for the cofactor for your wt and mutants. You can also determine the Kd for the cofactor (fluorescence, ITC...). This will also tell you or even confirm that these mutations have an impact for the affinity of your cofactor.

At last, if you do think it is a bit "fun", you can check the influence of these mutations on the activation parameters by doing some kinetics (binding of the cofactor). Also, as you do have a cofactor, does it help during catalysis (I guess so). So check the intermediates you are forming during catalysis (kinetics again) and you might then determine if you rate-limiting step, kinetic parameters (simulation program might help) are affected.

Dear Ismail Bustos-Jaimes. Thank u for your reponse. Yea probably that could be one Possibility,if the mutation brought would be at residue which is not catalytically actively involved. but how come the formation of active ternary complex is possible or would suceed when the actively involved residue in catalysis is impaired..?!

If you are purifying the mutant enzymes of the same columns as active enzymes, it is possible to carry some active enzyme over from previous purifications. Is the amount of activity within experimental error? If it is a hydrolase, perhaps water is substituting as a nucleophile. Otherwise, perhaps your postulate mechanism and/or nuclephile is wrong.

This is a difficult to answer in a few lines, but obviously you can obtain a lot information of the comparison. However, I do not understand why you change the assay conditions. If you change that, the comparison is not so useful and much more difficult.

Usually, change amino acids at the active site decreases the enzymatic efficiency (kcat/Km) assuming that evolution have optimize the active site. But this is not always true, and you can find in the literature hundreds of examples where "mutated enzymes" act as catalysts even better that the wild enzyme.

Are you absolutely sure that the residue you have mutated is, in fact, the essential catalyti residue? It could be near the site of catalysis, but not intimately involved in the catalytic chemical event. Second, how much did you have to increase the amount of mutated enzyme to get measurable catalytic activity? If it is a lot more, then you may be seeing a catalytic event that is not coming from the active site area. There is literature on the observation of catalysis coming from reaction of surface Ser residues, for example. You have not specified what type of reaction you are studying. What is the chemistry of the substrate cleavage? Are you used an "activated" substrate, such as a para-nitro anilide or active ester substrate?

Dear Irshad,

This question has two interesting layers.

The first one is somewhat trivial (even though interesting because very few people usually think in these terms), the chemical activity is commensurate with time. In another words if you wait long enough (sometimes longer than the age of the entire universe) the chemical reaction would proceed without catalysis (in gas or liquid phase). What it means is that everything (including your "inactive" mutants have "some" activity) and has some rate (sometimes infinitesimally small). So there is no qualitative difference between your "active" and "inactive" enzymes and your duty as a researcher is to establish the level of activity and differentaite it from the base reactivity (without a catalyst). The two simplest variables to change are protein and substrates concentrations. To illustrate this point I am citing two papers from my past where the activity was knocked down by multi-thousands fold (J. Mol. Biol.. 277(1998)647, Acta Cryst D 61(2005)1072).

The second layer is much more exciting. This is a question about the details of a catalytic process and what happens in the case of a mutant. In order to answer this question we have to have a rudimentary understanding how a particular enzyme (and as a matter of fact, every enzyme) is working. Without the detailed knowledge of the enzyme I cannot help you in details but I can point you to the important factors. Biological catalysts (that includes proteins, nucleic acids or even lipids) work by accelerating the reaction taking advantage of several physical processes that are contributing to what is commonly described as a transition state stabilization, or equivalently, lowering of the crossing thermodynamic barrier. These processes are:

1) Detailed three dimensional alignments of the incoming reactive atoms of the substrate and the aiding groups of the catalyst (residues of the protein). That applies to the angles, distances, and planes as the orbitals must be disturbed to achieve the final electronic transition leading to the formation or breakage of the bonds.

2) There must be detailed alignments of the charges (or charged groups of the substrate and the catalyst) to facilitate the electron or proton transfer responsible for the realization of the chemical rearrangement. Therefore the active site groups and pH play a crucial role in activity.

3) There must be substantial change in the electrostatic field at the active site to promote the reaction. This is why most of the active sites are located deeply in the protein matrix. By closing active site loops and expelling solvent (water) the dielectric of the medium changes by a factor of 20 making any electrostatic interactions stronger by the same factor thus facilitating the chemical rearrangement which is nothing else but changes in electron distributions.

4) What directly follows from the last two points is that enzymes usually must isolate the active sites from the aqueous environment by closing the loops and expelling mobile waters.

5) The act of catalysis itself is not static and definitely not an equilibrium event (as considered as an individual act), therefore, it is a dynamic nonequilibrium event. The height of the barrier preventing spontaneous chemical transformation varies all the time as well as the velocity of incoming substrate. Therefore, the protein dynamics plays a crucial role in enhancing the fluctuations leading directly to the enhanced rate of crossing the barrier. Almost precisely like a stronger gun promotes shooting through the shielding.

There are several other factors influencing activity but let's focus on the presented above.

In answering your question why your "inactive" mutants are active you have to determine which of these factors are changed, and which are preserved in your mutant, and which are playing a crucial role in the acceleration of the rate. It is obvious that almost everything stays the same, with the exception of the positioning of the crucial oxygen atom that was located at the Glu residue of the WT enzyme. So the answer is probably covered by the experiments that I cited. When you exchanged Glu for Asp you deviated the distance between the substrate and the crucial oxygen by at least 1 Å. Such changes usually lead to a change of the rate by 100 fold. In changing Glu to Gln or Asp you probably changed the polarizability of the oxygen in question (pKa) by at least several pH units which leads to changes on the scale between 100-1000 fold. In Ala mutation probably a spurious water molecule took over the role of a reactive oxygen and lowered the rate by more than 1000 times.

In conclusion there is nothing surprising that your mutants have activity and that you were forced to raise significantly the concentration to see it. Concentration is usually a proxy for changing the time or energy state of your system to access the areas that are not available under "normal conditions". This is like rate of nucleosynthesis that is not normally observed (it would constitute a medieval transmutation in alchemy): you have to have much higher pressure and temperature to get to force the nucleons to coalesce and form new atoms. So we can quite easily transmutate nowdays the elements into each other in nuclear reactors.

I hope the answer was not too long so I do not loose the audience but I think that many similar questions are posed on the Research Gate and do not get in depth response.

To provide a support for your results for the publishing purposes you just simply search the literature and find similar cases. More mutations or different assays might but not necessarily elucidate any possible answer.

Dear Irshad Baig. It is difficult to answer your question when no more data is available (to me), e.g. which residues you mutate and how those residues are involved in catalysis. Maybe you overestimated the role of a single residue in catalysis or the new residue, in some way, reproduce the role of the original residue.

It is clear!- This question has a lot points of view. All comments are interesting and right but as Lasse said above, some of them can increase the confusion about so general question. Cheers everyone.

I would have to disagree with Lasse and Francisco last statements. In the last 5-7 years a substantial progress have been made in understanding enzymatic protein activity. Starting from simple predictions of the active site locations from the structure or even sequence alone by advanced bioinformatics programs, to producing de novo enzymes (vide D. Baker), or to describing the features of an effective enzymes (Kern from Brandeis) by studying protein dynamics. Allostery has been tackled in a very comprehensive manner by a number of distinguished researchers.

By knowing the very rough details of substrates and the general features of the particular enzymes we are able to propose nowadays the details of chemical reactivity. I am not even mentioning in this context detailed correlations in molecular beam techniques between different elements of reactivity and the quantum mechanical description of these properties.

Additionally, the question Irshad posed was quite specific. In essence he asked: Why my expectations are different from what I find in the experiment. In my answer above, I pointed out that his expectations were incorrect. Forming our scientific intuitions is much more important than answering an individual questions. Every scientist is bound to make an incorrect statement at least once in the life time so the general knowledge is much more important, and this is why we use ResearchGate.

Another possibility to rule out, depending on how much lower the activity is, would be presumed infidelity on transcription / translation leading back to a very small percentage of wild-type being present. (i.e. does the small amount of activity vary upon changing the codon in question to another codon for the same residue type.)

Other people already give you many scenarios, but one thing you need to figure out is if your mutant enzyme is produced in a heterogenous host such as E coli and if the E coli also poses such enzyme resembling that activity. If the answer is yes, there is a good chance that you may have co-purified the host enzyme along with your recombinant one. Because the level of copurification is low, it's like contamination, so the activity you can detect is rather low. It is easy to rule this out by performing a substrate affinity assay. If the activity is due to contamination of host protein, the Km should be the same as that (reported) to the host enzyme, but the activity is due to mutant enzyme, the Km is ALWAYS higher than that reported for wild type enzyme.

If you can provide more data, more insight can be given.

Best

By the way, say hello to Professor Yoon for me :D

Dear Stefan Walter Sir, Thank you for your valuable suggestion and the possibilities you raised.The residue i targeted is reported to play key role in cofactor binding and thereby leads to catalysis.The residue is not involved in substrate binding (actively).Hence,reduction in Kcat for mutant could be justified,however marked increased in thier Km,is not understood to me Yea, i do agree with your view,possibly some activity could be from adjacent or any other residue of the enzyme which is not been reported yet(as i know). Therefore, to rule out this possibility, i also studying few highly conserved residue,which lies in the immediate viscinity of this catalytic residue. Hopely,soon i could able to figure out thier active or supporting role. Thanks.

Mainly Boguslaw Stec , J.Friedman Dung Le gave you very good detailed explanations / suggestions / advice and there is little to add. I just like to iterate in very simplified terms, you have to distinguish between the residues who are mainly involved in binding (determining the lifetime of the central enzyme-substrate(s) complex ) and those who are primarily carrying out the catalysis, the formation or hydrolysis of a bond etc, Look up the old classic discussion by Monod of the very basic differences in enzyme mechanisms --most enzymes are between his formal models. Then do a exhausting literature search --not only the last 3-5 years-- on your enzyme reaction, find the suggested or known mechanisms for your reaction in the literature and see with what you are dealing. As said by other very specific suggestions can not be made without knowing exactly what enzyme reaction you are studying and what the numerical differences are. I am ot all surprised that you found minor activity with your mutants for reasons others above have eluded on.

Dear Dominique Padovani Sir, Thank you so much for your time taking deliver to me your valuable suggestion/guidance. Your explanation regarding the change/mutant residues is convincing and satisfactory to me(as i too considered this facts).Further,i would go through more literature survey in related enzymes, to find if the mutant residues would affect any intermediate during catalysis (directly or indirectly) Besides,i would certainly take your advice and carry out further experiment related to enzyme stability and binding affinity determination using ap enzyme,as you rightly suggested,and as i feel it would be quite essential to prove the role of my target residue. Since, iam not well-versed with this techniques,i would certainly in need of your precious guidance through email(if you provide) to accomplish the task. Hope,you would surely extend your valuable guidance to me in this regard. Thanks again.

It is difficult to know without knowing the enzyme, but some general aspects are discussed in Peracchi, 2001, Trends in Biochemical Sciences 26 (8) 497-503 (and there are probably more recent reviews). Basically, most enzymes are designed to decrease the activation energy by more than one mechanism, so having eliminated one aspect of catalysis may still leave an enzyme with (albeit much lower) activity. However, there are definitely more trivial reasons, such as contamination of the mutant enzyme with wildtype enzyme. For instance, we recently found that our glycosynthase had about 0.1% of wild type hydrolase activity due to contamination with wild type enzyme, which evidently came from either mismatching of the tRNA at the ribosome or reversion mutations in the expression culture (we did not differentiate, but there are references for both types), since we did not use any purification resins or other supplies used with the wild type enzyme. Adding a wild type-specific mechanism-based inhibitor or changing our mutation from one requiring a single base pair reversion or mismatch to one requiring two decreased the wild type activity to an acceptable level (Pengthaisong et al. 2012, Carbohydrate Research).

Hi Irshad,

my e-mail is in my profile --> info.

For information - For the cofactor affinity I guess you can perform the same experiments than those realized in your paper, i.e. by fluorescence. If your Kds are "high", only fluorescence might work and you will not be able to use ITC. Otherwise, you can also determine a "kinetic Kd" (kon, koff) by stopped-flow. If your cofactor absorbs and its binding does affect its spectral characteristics (SF UV-vis) or change the A280nm of your enyme or the fluo of your cofactor (SF fluorescence).

For the Tm, it is quite simple: increase gradually the temperature of the buffer+enzyme and follow the F340 nm (excitation at 280-290 nm). The temperature range will depend on the Tm but usually you can go from 5oC to 60oC. Otherwise, you can use DSC (Differential Scanning Calorimetry) if you do have this instrument around.

Also, I do agree with some comments in here, i.e. it is often essential to check the activity as a function of ionic strength or pH, especially when you consider having some charged residues in your active site!

Thanks to all for your valuable suggestions and advice to me. I apologise sincerely for my bit delayed response to each of you.

Dear Brock Schuman, Thanks for giving your point of view and to raise doubts about the posisble experimental error. Although, I do extensive washing treatment before each new purification (Ni-NTA /Gel filteration G-superdex 200 /MonoQ ,various coloumn, I employed). I too realized the possibility you raised,that some active (wild ) enzyme could be co-purified during purification of mutant enzyme. However, if this experimental error, why do is only incase with mutant? The property of mutant (low activity) could also be acuired by the wild enzyme,however its not. The wild enzyme found to be equally active irrespective of times,I purify.

Comparatively,since the kinetic parameter ( Km) is quite lower and (Kcat) is higher by 1000 fold of wild enzyme,thus I questioned myself? If mutant enzyme is somehow contaminated by even trace amount of wild enzyme,why do I observed lag in product formation even at higher higher substrate level. The kinetic paramter for Wild enzyme (Km) 2.5mM, mutant reflects activity at over 500-1000 mM.

In brief, I interrogate myself, if the wild type is get co-purified with the mutant enzyme,why do it looses its normal kinetic behaviour?

Dear Francisco Sir, Thanks for responding to my query. I did (had to )change the assay conditions (optimized to conditions) just with a curosity,whether why only mutant reflects no activity, where as my wild enzyme do reflect,hence I began thinking,if is reported in the literature,if the residue is critically important (in cofactor binding) ? Would the mutant have any impact on increasing that cofactor or substrate, and I found my little postulate worked out. Possibly, my approach or understanding is incorrect in dealing or studying mutant enzyme ,hence I look forward to hear valauable advices from you all above distinguished research scholars on this aspect . Hope to get lot more valuable suggestions. Thanks.

Kind regards,

Irshad.

Dear Lasse Greiner Sir,

Thanks a lot for your valauable suggestion to my research query. Ido appreciate and agree with your view, that since I do change the charged (catalytic) residue,I must also study the mutant enzyme kinetic behaviour, under various conditions of ionic strength, array of buffer, pH, temperature. I would do further study to gain more actual insights. Thanks again.

Sir, thank you for your comment on my post. I apologize that,I did not provide the details of the enzyme . I study biosynthetic enzyme called AHAS (acetohydroxyacid synthase ;plays a role in BCAA’s synthesis) belonging to of class ThDP dependent enzyme family. AHAS catalyzes condensation of two molecules of pyruvate (substrate) to form acetolactate or acetohydroxybutyrate,which is further converted to BCAA(valine,leucine,isoleucine) by other respective enzymes of the BCAA pathway.

Yea sir, iam sure the residue, I focussing is involved in ThDP binding and its activation,which further leads to its catalytic action.

I do have to make a increment of 5-10 times in the amount of mutated enzyme to obtain measurable activity. Sir, i have few doubts to get clarified from your expertise. Although,I do increase the amount of enzyme(mutant) to see the activity. What let the mutant enzyme show activity(although lower than wild enzyme) at higher enzyme cocentration?. Is there a possibility, that part of the mutant enzyme being expressed and purified is inactive and part of it remains active?. Would there be some way to rule out this particular possibility.

I often self interrogate, is my approach of increasing mutant enzyme concn in an assay to see some activity is correct or has some scientific base or just my trivial appraoch.? As a learner,I could not able to get the answers on my own. I hope,you would lend or shed some light and your valuable approach in this regard. Thanks again

Dear Boguslaw Stec Sir,

Its indeed interesting to read your lengthy and valuable suggestion and advices bestowed upon my research query.I do agree with the reasons you give which are essential in accelarating the rate of reaction by biocatalyst and for the mutant enzyme. As suggested,I have refered to your cited refrences and finding it helpful. The answers you gave is quite satisfactory to me,hope you would extend your valauable guidance to me in future too. I also thankful to you,since you understood well my situation and appreciated my research query. Sir, although you have indicated in your comment,that more stringent conditions can sometimes be needed to complete the reaction.I still would like to ask you few my doubts and get your valuable advice.

Before that, n brief I would like to mention that I study biosynthetic enzyme called AHAS (acetohydroxyacid synthase ;plays a role in BCAA’s synthesis) belonging to of class ThDP dependent enzyme family. AHAS catalyzes condensation of two molecules of pyruvate (substrate) to form acetolactate or acetohydroxybutyrate,which is further converted to BCAA(valine,leucine,isoleucine) by other respective enzymes of the BCAA pathway.

I study the role of some highly conserved residues. The important features include,

It lies in the active site region of enzyme. One of the residue (Glu) is believed to be involved in ThDP binding and its activation,which further leads to its catalysis(reported by many researchers).

Sir, i have few doubts to get clarified from your expertise.

I do have to make a increment of 5-10 times in the amount of mutated enzyme to obtain measurable activity. Although,I do increase the amount of enzyme(mutant) to see the activity. What let the mutant enzyme show activity(although lower than wild enzyme) at higher enzyme cocentration?. Is there a possibility, that part of the mutant enzyme being expressed and purified is inactive and part of it remains active.? Would there be some way to rule out this particular possibility.

I often self interrogate, is my approach of increasing mutant enzyme concn in an assay to see some activity is correct or has some scientific base or just my trivial appraoch.? How would I able to support it to the reviewers,if asked? although trivial, am I drawing correct conclusion or my interpretaion is incorrect which perhaps would mislead other researchers. Sir, I apologise for being more expressive, however I personally felt that you would understand that perhaps due to my infancy state of knowledge in enzymology,I could not able to get the answers on my own. I hope,you would shed some light and lend me your valuable suggestions.

Thanks.

Kind regards,

Irshad

Dear Sir (Jonathan Friedman). Thanks for your valuable comment and the possibility you highlighted. Yea probably,the possibity you raised could be possible. It seems,the same possibility, Sir Boguslaw Stec had rule out with his interest of mutant protein (as cited in his reseach refrence, Acta Cryst D 61(2005)1072). I would certainly take your advice and work out, if there is any significant effect imparts on mutant enzyme activity by changing possible codons. Thank you.

Dear Sir (Dung Tien Le),

Thanks a lot for your valauble suggestion and advice. I would certainly work on it and would pleased to provide more needed experimental data and get more valuable insight.

Sir, yes I do express my protein in heterogenous host such as E. coli (Bl21) and there might be a possibility as you mentioned,little amount of wild enzyme get co-purified along with the mutant enzyme. I would surely try to rule out this possibility by growing simply E. coli (Bl21) without harboring mutant and following same procedure.

However, I wonder ,if our this possibility of the presence of small amount wild enzyme is taken for acceptance,I have performed substrate afinity test several times with the mutants and I found the Km of in the assay is around 500-1000 folds higher; it never reflects any kinectic property of wild enzyme.

Anyway,I would convey your wishes and keep in touch to get your valuable guidance. Thanks.

Dear Stefan Walter Sir, Thank you for your valuable suggestion and the possibilities you raised.The residue i targeted is reported to play key role in cofactor binding and thereby leads to catalysis.The residue is not involved in substrate binding (actively).Hence,reduction in Kcat for mutant could be justified,however marked increased in thier Km,is not understood to me Yea, i do agree with your view,possibly some activity could be from adjacent or any other residue of the enzyme which is not been reported yet(as i know). Therefore, to rule out this possibility, i also studying few highly conserved residue,which lies in the immediate viscinity of this catalytic residue. Hopely,soon i could able to figure out thier active or supporting role. Thanks.

Dear Sir (Juergen Wiegel)

I do agree with you, I received many valuable suggestions and guidance from you all distinguised researchers. It really adds to my knowledge. I would surely take your advices you given in your comment and would work out accordingly. Thanks Sir.

Dear Sir (James Ketudat Cairns)

Thank you for your valuable suggestion and providing possible reasons. I would surely look into it and try to rule out the possibilities you mentioned in your comment. I have acessed your cited article,I would soon be refering to it. Thanks Sir.

Irshad,

I agree with you that the fact that the Km and Kd have changed suggests that the new activity is most likely due to the activity intrinsic to the mutants and not due to some residual native protein left from the expression system (although there could be a small amount of wildtype contributing along with the mutants, which is hard to completely rule out unless the wildtype has a clearly different activity from the mutants). Give this, I think the residual activity is more likely due to the factors pointed out by Boguslaw Stec and the review that I mentioned in my previous answer. Obviously, if there is contamination with wildtype, you would get two activities contributing, which would show up in a v vs. v/s plot, if the wildtype is significant (hopefully, it is not). Regardless, you are left with the more interesting challenge of discerning what has changed in the mechanism of the mutants.

As an aside, the contamination with wildtype from the expression system (if not from the purification) is highly dependent on the cells used for expression and the exact codon change that is made. We compared the same change in catalytic residues in two different proteins expressed in different E. coli strains due to their different requirements for activity and found that one had no detectable wildtype contamination at any level, but the other with the same mutation did.

I remember that there was a paper by John Gerlt a few years back in either Chemical Reviews or Accounts of Chemical Research that outlined some of these possibilities.

I thank James Cairns for bringing up a review I wrote on this topic years ago... Indeed, if the Km (or Ki for some specific inhibitor) has changed significantly, it seems reasonable to assume that the activity you detect is not due to wt contamination. Other contaminations remain possible (partial revertants, unrelated enzymes from the expression system....) but you should be able to sort out most of them by carrying out a rigorous kinetic characterization of your enzyme preps. For example, contamination by a host enzyme should be reasonably constant in all your preps (provided that all mutants are purified the same way). So, if you got roughly the same residual activity for all your mutants, I'd be very suspicious...

The fact that you can observe activity by the mutants only by 'forcing' conditions (high S, high enzyme) is not at all in contrast with the hypothesis of your mutant(s) retaining some intrinsic activity. The arguments put forward by Stec and by others in this forum are impeccable. If a mutant retains some feeble activity, the sensitivity of your 'standard' assay could be simply insufficient to detect it.

One further, quick check I would do is making sure that, under the conditions where you can measure activity, the rate of the reaction is directly proportional to the concentration of the enzyme (I mean, if you quadruple the enzyme conc, activity also increases fourfold). Since acetohydroxyacid synthase is an oligomer, a mutation could in principle shift the monomer-oligomer equilibrium...

If the residue is involved in binding with co-factor, you have to tell us which one, as I know AHAS, i assume it is the enzyme you are working now, has an FAD, ThDP and Mg2+ as co-factors, and among these, FAD actually does not involved in catalysis. The requirement of this co-factor have been discussed elsewhere, mostly for structural integrity.

If your residue of mutation is involved in interacting with this co-factor, your mutant enzyme may still work, and even as robust as the wild type one, depending on which aminoacid it was replaced to.

In this enzyme, the active site is formed on the interacting surface of the homo-dimer. There also another possibility that there are some dimer where one subunit is your mutant, while the other one is E coli host catalytic subunit and this hybrid enzyme can give you some residual activity.

If you still have not satisfied with all the explanations, send me your data via your advisor, Prof. Yoon, and I will take a closer look at it for you.

In addition to all the possibilities commented above, you might also consider an aspect commonly found in discussions related to the evolution of novel enzyme activities in nature.

Through evolution, genes might get duplicated or, due to other mutations, loose they purpose. This open the door for mutations leading to new activities/functions for the protein.

Some data (and yours would be an example) suggests that enzymes have evolved to have "alternative" catalytic mechanisms, which would stay hidden because the "main" function is remarkably efficient. When mutations occur, this alternative mechanisms are disclosed and you may observe a low but measurable catalytic rate, even when some "essential" catalytic residue has been mutated for a completely different one. In addition, other catalytic activities may sometimes be detected (a situation known as enzyme promiscuity).

In the end, though apparently high in diversity, enzyme catalytic mechanisms can be grouped into a number of succinct categories (see Gutteridge A & Thornton JM (2005) Understanding nature's catalytic toolkit. Trends Biochem Sci 30(11):622-9. ).

You may want to look at this review, which i found very interesting Khersonsky O & Tawfik DS (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem 79:471-505.

Best wishes,

Rogelio

In deed, worthy discussions were made in reference to 'Enzymology'.

You said "I found that the mutant reflects no activity at the normal identical assay condition provided for wild enzyme. However, when the substrate cofactor and enzyme concentration is being increased in an assay, the active site residue mutants reflect measurable activity, although less compared to wild enzyme.The mutant enzymes have demonstrated less affinity for substrate and cofators since the kinetic parameters like Km and Kc values have increased by several fold"

Again you said ".Would it be appropriate to compare the kinetic results of mutant with wild enzyme? The assay conditions vary in each case. "

Already you have done something very sincerly and honestly but you are contradicting yourself about the experimental setup eith the mutant and the wild variety.

What ever may be the situationresults of or confusion, it is most appropiate to compare the kinetic results of both the types, and you have already done it.

However, your main problen is, how the increased cofactor concentration is bringing some amount of activity in the mutant types? OK, your mutations are, Glu to Ala, Asp, Gln, Asn and I hope,the Asp mutant is showing highest activity among the mutant enzymes. Rest, you apply structural proteomics and functional bioinformatics, you will get the satisfactory explaination for your findings and observations for proper interpritation. Excellent work, just make it tidy and systematic for the finishing touch.

Dear Irshad

I would like to know what you made out of all the answers you got. Some mad some unlikely explanation, others gave you true but sophisticated answers. What did really helped you to tidy down your explanation. I got redirected to your question by being notified that a new answer was given.

I would also like to reiterate  the very basic explanation going back to the fundamental enzyme  reaction, which is true for all enzyme --as far as I know-- regardless whether it is a simple 'one Substrate - one product reaction' (where you can use the simple Lineweaver Burk) to multi substrate-multiproduct-multi subunit enzyme (where you have much more kinetics i.e., as shown in Irvin Segel's Enzyme kinetics) see some comment  further below).  Every enzyme (biological catalyst) is in principle reversible)

[E] + [S1,S2, S2]   [E-S1-S2-S3] (called quaternary S complex) [E-Pn] [E] + [Pn]. How many steps of S binding and P released  and which binds first  and which is released first etc. depends on the Substrate binding product released mechanism (See Clelands nomenclature on this).

themain point is: The resulting enzyme reaction is in principle depending on the stability  of the various complexes (or simply spoken affinity of the enzyme for any Substrate / ligand). In short the  Km-values are a reflection how stable the Enzym-Substrate and Enzyme-Products complexes are and whether they exist long enough to form the E-Pn complex and  release the Product(s). The later one is usually not the time dependent step like the formation for the Enzyme-Substrate  and the Enzyme Product complex -the latter one also called the  catalytic step and usually the velocity determining step. (a slight variation occurs in the Ping-Pong mechanisms).

I you change any of the aa side chains in the catalytic center  (or one in the protein which causes a change in the Enzyme structure -- or if applicable for the allosteric center or the multi subunit structure; also pointed out by previous respondents))-- the binding of  one or of several  ligands  (including co-factors and the ligand which are not getting changed but are required for the correct structure for catalysis {see Monod's model of the principle enzyme  reaction and allosteric situations} will change usually to less binding, i.e the back reaction is faster then the catalysis. By increasing the ligand concentration you increase interactions of the ligand(s) with the enzyme and thus favor the interaction of the ligand with the enzyme and thus the possibility that one of the Enzyme - Ligand complex is more stable and long living enough that the catalysis occurs.

All the other suggestions are in my opinion secondary for a simple justification why you get good activity at higher substrate (Ligand)  concentration

One other point I would like to make because as a reviewer I see too often that a the following mistake is made when dealing with multi Substrate-multi Product and Enzyme reactions where an  Enzyme metal complex with an easy disfusible metal is involved.  

--I assume you did not use for the Km determination Lineweaver -Burk, i.e., by varying one substrate and hold the others constant and used simply the intersection for calculating 1/Km, but did the permutation of varying one substrate, i.e. varying ligand a in a series of various  (at least 5 each)  of varied constant  ligand B  and  keeping ligand C constant. (this yield five lines of 1/v vs 1/S). Than you have to do the same but do the permutation for  A, B and C. then you do the corresponding replots depending on the enzyme binding modus e.g., according to Cleland's nomenclature. 

I hope you enjoy my points even if you have solved your problem already to satisfaction of your adviser because enzyme kinetics are fun, although in many Universities in the molecular and genomic age  very little or not at all taught beyond MM-kinetics

 Dear Prof. Juergen Wiegel,

Thank you so much again for remembering and adding more explanation here. Yes, I got valuable suggestions from many good expertise in this field and it really helped me to think in many possible ways and gain good knowledge.

Now, when i looked back, i think, as a beginner without much literature research survey of related enzymes, I filled with so many questions and curosity that how a single point mutation at particular site of enzyme leads to inactivation of enzyme, where as the same wild-type enzyme shows the enhanced product forming effect. So, i decided, before I  conclude that the various constructed enzyme mutants were inactive, i wanted to test at various higher concentration of enzyme and substrate and cofactors and see, if it make some difference assuming that the requirement of mutant enzyme could have been differed upon mutation (just my blind assumption). Co-incidently, yes it worked out, although very very less (compared to wild-type), but i see a detectable or measurable activity upon increased substrate concentration and cofactors in mutant enzymes. More interestingly, the increased of activity in mutant followed the same rule of side-chain chemistry, as also same mentioned here previouslyin the comments. Briefly, the determined kinetic parameter, kcat for wild type and mutant enzyme are as follows i.e Glu (3.196) >Asp (0.0712)>Gln ( 0.0384)>Ala (0.0026).  I explain this results in my paper and to reviewers with the explanation that due to lack or short hydrogen bond distance between this crucial Glu (-COOH) and N1 atom of ThDP the mutant reflected lesser activity and supported it through molecular dynamics simulation approach. Not only here, but also, a point mutation at another site of the same enzyme, where i replaced a conserved Proline to simple (Ala), hydrophobic(Val), hydrophilic (Thr) and acidic (Glu), all except one mutant  reflected activity but with decrease affinity for substrate. However, at a certain elevated level of its essential cofactor (ThDP) concentration, this mutant((Pro-Glu) ) reflected activity.  I understand, that with a chemical perspective, this can be easier fact that a completely opposite Glu residue would have loss of activity, but my real query, if all that stands true, why a elevated level of ThDP reflected activity? Unfortunately, with not great appreciation and encouragement, i could not go into depth of this aspects to satisfy my queries.

But, what i all got to learn through  this enzyme active site mutant study is that increasing substrate or cofactors or studying the mutant enzyme at different assay conditions than wild-type may not be a right approach (since many of the researchers study or reccommend to measure the effect of mutation at similar assay conditions for both wild-type and mutant enzyme).

But, another thing, which i learned and similar to your opinion about Monod's model of the principle enzyme reaction that although short-lived enzymatic state, but  a increased substrate or cofactor ligand concentration might bring some local and favorable structural conformational change which try to bring enzyme's other similar functional residues into correct geometrical position for catalysis. However, i could not able to experimentally prove this observation to be true, but if i get an opportunity further under great expertise, I would like to explore this aspect in depth and find out if my observation was true in some sense for other enzymes too. I agree as you said, these days not many appreciate or encourage to go into depth of basic sicnences enzyme kinetics.

I really enjoyed discussion on this topic and noted down many important kinetic aspects which I certainly going to keep in my mind and apply in my further research.

Thanks to all of us here who spent their valuable time and extended me their valuable suggestion and opinion, which really helped me to gain great insight.

Sincerely,

Irshad Baig