The pH dependence is usually due to the side groups of the amino acids. A change in pH changes the protonation pattern and can, in extreme cases, result in protein denaturation. I therefore suspect that your protein stabilizes the enzyme structure, thus keeping it active at sub optimal pH values.

The pH dependence is usually due to the side groups of the amino acids. A change in pH changes the protonation pattern and can, in extreme cases, result in protein denaturation. I therefore suspect that your protein stabilizes the enzyme structure, thus keeping it active at sub optimal pH values.

The answers above are correct. I could reiterate but why bother? :)

Just to PS1 - in vitro enzymatic assays are important, however you should be careful in interpreting them from an in vivo perspective, as there are many other factors that you should consider that you just cannot replicate in an in vitro system, such as membrane interactions and protein-protein interactions that are not known.

Which kind of enzyme are we talking about? A change of pH can destabilize enzyme structure, or protonation of residues in the active site involved in the ES complex stabilization or in the catalysis, and finally pH can also change the protonation of substrate if this has a pK in the range of between 5 and 7. The presence of a protein can stabilize enzyme structure of substrate protonation. If you are interested in the study of the effect of pH on your protein you can consult a good enzymology test such as: Structure and mechanisms in protein sciences. by Fersht A.

As pH interferes with side groups of proteins that can be protonated/deprotonated (see above), the additional protein could act in two ways. 1. It could directly interfere with the enzyme in question and protecting certain side groups from protonation/deprotonation. 2. The protein could act as a "buffer" by being protonated/deprotonated before the enzyme.

Thanks all for your comments.

I know different pHs would change the protonated status of the side chains of the aa, but making my question a little bit more focused (actually 2 questions):

- How is a second protein overriding the protonated/deprotonated status of my enzyme? For this question I like Horst Onken first hypothesis, not the second though because I used BSA (in excess to rule that out). The flaws of that hypothesis would be: if it's protecting the active site, why is the enzyme working better even though there's a protein blocking-interacting with the active site? If it's not protecting the active site, my enzyme could/should have a regulatory domain ... not coming from a BLAST of the sequence (no homology with regulatory domains). But this is maybe a nice hypothesis.

-The second and almost more important is: do you know any similar, already reported, effect? Any papers with similar findings? I'm just worried this could be an artifact because I can't see the in vivo relevance of this effect (better functioning of the enzyme in different organelles with different pHs?).

Thanks again!

I don't think anyone else has mentioned that between pH 7.5 (high activity) and pH 6.5 (low activity) you have the pKa for the sidechain of Histidine. Its quite possible that histidine is part of the enzymatic process of the protein. Of course it is also true that significant structural changes have occurred because of the pH change. Also, if you have changed the pH by direct addition of concentrated acid then this can mess with the protein greatly in a way that it can not recover from.

I'd be interested to know if you really see a ten-fold drop in activity from pH 7.5 to pH 6.5. This pH change would have to be made by dialysis so the change is not too dramatic. If you still see the change in activity and there is no major change in the structure of the protein (which could be tested with low resolution by running the protein over gel filtration and see if retention time changes, or maybe by looking at it's CD spectrum) then I would start to believe that one of the active residues is a histidine.

Your control with BSA does indeed seem to rule out that the additional protein is just buffering. It appears the mechanism you observe is more complex.

I do not know your enzyme, and I do not know the protein that changes its pH optimum. However, an inhibitory effect of protonation/deprotonation must not necessarily be related directly to the active center of the enzyme. The effect of pH at some place of the enzyme could just change its conformation and affect the active center indirectly. If your additional protein would interact with your enzyme in a way that the protonation/deprotonation and or the conformation change is prevented, then you could observe less sensitivity towards pH changes.

I do not know of literature relating to the topic, but I could imagine that it could be instructive to explore on a molecular basis why pepsin has a pH optimum in a very acidic range, whereas trypsin has a pH optimum in a neutral or a little basic pH range. How is pepsin "protected" from denaturing at pH 2? Of course, in the case of pepsin the answer lies in the structure of pepsin itself. However, the additional protein in the case of your enzyme may mimic the surface of pepsin. Because pepsin and trypsin are very well-studied enzymes, I could well imagine that there are references to this.

I found this article on how residue pka values may be perturbed by protein structure and I thought it might interest you. I don't think that you identified the enzyme and this might be useful information.

If you want to know if it is a specific interaction, or just a molecular-crowding like stabilization, you can try adding some lactose to your buffer system at lower pH, since lactose has the same molecular-crowding effect, often enhancing enzyme stability in high temperature or non-optimal pH values.

Looking at your results, I could imagine several "crude" explanations : pH-dependence may be related to two major things : 1) pH modifies the protonation states of amino acid residues side chains, which in turn modifies your enzyme's state (structure, etc...).This could eventually trigger denaturation, at first, and lead to a loss of activity. 2) If your the catalytic mechanism of your enzyme involves an (or several) amino acid(s) residue(s) that is (are) involved in an acid/base mechanism, then changing the pH changes its protonation state and inhibits/enhances (depending on the pH) its catalytic mechanism.

You could interpret it in two ways (one of thelm being of scientifical interest, the other one being just an experimental artifact) :

If you do add another protein to your assay, you may "buffer" the pH because it can "absorb" protons and therefore may protect your enzyme indirectly, just as a true buffer would. However, since BSA doesn't seem to have the same effect, it might be possible that your secondary protein could stabilize your enzyme, as, for example, a chaperone would do. This would explain your results.

To decide between those hypothesis, you first have to be sure that your secondary protein (putative chaperone or interactant) is dissolved in THE SAME buffer as the one you're using for your enzymatic analysis, or the kinetic recovery could be interpreted as an effect of your secondary protein's own buffer (that would turn back your pH to a more "suitable" value). If you suspect any interaction or complex formation between your enzyme and this protein, you can't forget to demonstrate that a true interaction occurs in vitro (or in vivo). To do so, you have to use other techniques, as for example, retardation gel shift assays, size exclusion chromatography (for non-equilibrium techniques) or isothermal titration calorimetry (ITC), thermophoresis, biacore, and so on (for equilibrium techniques, that can give you better affinity constants, since your truly are at the equilibrium). In vivo techniques such as co-immunoprecipitation or (bacterial or yeast) two-hybrid could also be used together with FRET approaches.

Well, hope you'll find some ideas to overcome this problem.

There is one other option that I do not think has been considered. It may be the second protein is actually improving the delivery of your substrate tot the active site. If the second protein binds the substrate, then this may mean it is acting like a chaperone of some sort. This does happen in degradation processes and in incorporation of metals into active sites. such as iron into transferrin.

Also consider the comments about the pKa given above, especially for histamine. Changing the pH will alter the higher order structure of the enzyme and the second one differently. How this alters the structure may be relevant. Finally, BSA may not be the best negative control. Can you work out the isoelectric point of your second protein and then use another protein with a similar Pi as a "charge negative" control?

Good luck

The charge of the aminoacids at the active site of the enyme are highly influnced by pH. Aminoacids are amphoteric , the charge they carry is pH dependent. The enzyme mentioned above seems to be active at slightly alkaline pH. At acidic pH the activity drops .Usually at a pH below the pI proteins carry a net positive charge and above pI they carry a net negative charge. The change in charges influences the binding interactions of the enzyme and its substrate, which influences the enzyme activity.

When mixed with another protein , the lowering of pH , might give suitable charges to the new protein which could mediate the interaction between the enyme and its substrate restoring the activity of the enzyme.

Enzyme activity does not solely dependant on pH and temperature, it depends on the substrate that you provide to the reaction mixture as well. May I know what is the enzyme that you're studying on? And the substrate used.

It is the nature of every protein to have their optimum pH because of different amino acid chains and structures that made up the protein itself. Since different amino acids have different pI (isoelectric point), combinations of different amino acids will give you different pI values when a protein is created. If you can still recall, pI is the pH where zwitterions are formed, and in terms of catalytic efficiency, pI is the pH where the protein/enzyme is at maximum stability and thus able to catalyse reaction(s) at its maximal velocity (Vmax). This is what we call the "pH optimum".

In short, if zwitterions formation occurred at the active site of the enzyme, that is the pH where maximal enzyme activity can be achieved. If is occurs at other site of enzyme, it might not be the pH optimum then.

Most of the theory has been covered above, however one fact was only superficially touched. Enzyme groups pKa are strongly dependent on the environment, of special importance are:

i) Hydrophobicity. In the non-polar interior of a protein the dielectric constant drops for 80, of pure water, to about 6. The charged form of amino acids becomes less favorable, for carboxilic acid residues, such as Glu, Asp or the C-term, this means an increase in the pKa. Increments of 1 pKa unit are common, but more dramatic changes can be observed. For the basic amino acids, such as Lys, His, the N-term, or Arg, this means a drop in the pKa.

ii) Electrostatic environment. Partial or formal charges in the vecinity of a group have an strong influence on its pKa. For instance, an Arg (usually charged) in the vecinity of an His would make more difficult for the His to protonate, because of the electrostatic repulsion, producing a drop in the pKa.

There are, as indicated by Horst, Michael, Sander, Frédéric, Harry, Malathi and Allan, several possible explanations to your observations. But one that has not been elaborated enough is:

Your protein active site might have different degree of solvent exposure when free, or when in complex with the interacting protein, either due to a change in conformation that impacts the active site, or because the binding site is close to the active site and modifies the accessible solvent area (ASA) of your active site.

You might get deeper into the aspects by analyzing the full Activity vs. pH profile of your protein as both saturating and subsaturating substrate concentrations, for both your protein alone, and the mixture. Plot the results an have a look at the Enzymes book of Dixon and Webb to see how you can analyze the data against pH to calculate pKa values. Be careful, an appropriate buffer selection is essential, and you nee to make sure to approach saturation with substrate at every pH, for apparent Km values are also pH dependent. Do not fall into the temptation ta ascribe a pKa to a particular amino acid, unless additional information is available, for as already said, pKa values can change significantly.

Best wishes,

It is very important to know the enzyme and its sustrate and the environmental pH where the enzyme is active. See for example acid phosphatase in prostate and alkaline phosphatase in liver. Your question must be more specific, because some times the enzyme depends on its catalytic site and the connection site with the sustrate, but also on the different distance between them, when the enzyme is activated or not. . Sorry, my answer is very simple but you have in this page a lot of contributions and I try to go to well known enzymes and their activation mechanism , because there are many differences among the huge number of enzymes or their groups. Many times we start to know one enzyme and it activation mechanism .Then, it is more easy to understand a given problem.

An enzyme is a protein and thus, has a large number of exposed amino acid side chains with a charge that depends on its pK and the pH of the solution in which they occur. This amino acid composition also means a specific form of folding of the enzyme protein and exhibit its catalytic zone. any change in pH, change ionozación side chains and consequently the folding of the enzyme protein and probably the accessibility of the substrate to the catalytic site.

The association with another protein can alter the expression of these charged residues and modify changes in the folding of the enzyme protein through protein-protein interactions, so that it is possible that the catalytic site is accessible for the substrate at a pH in that was not accessible to the native enzyme protein.

I think firstly you should told us about your enzyme and the protein used. The mechanism of dependence of enzyme activity on pH, will differ from enzymes class to another class. Oxidoreductases activity differed from pH value to another according to the substrate you used and it ranges from 3.0 in ABTS to 7.0 in case of Syringaldazine, and this depending on the oxidation state of substrate and the protonation between the OH and H ions. I think in your case is the stabilization of active site of your enzyme and saving the conformational structure of the enzyme.

Attached a paper illustrate the Effects of Redox Potential and Hydroxide Inhibition on the pH Activity Profile of Fungal Laccases.

This is usual phenomenon that enzyme activity depends on pH. Moreover, there are many multiple enzymes like hydrogenases which have high activity at different pH and inaddition they can reverse. Why? Enzyme conformational changes including active center, different interaction with substrate, presence of the other proteins (within the membrane) and protein-protein interaction etc. You should check different possibilities and show which is probable with your case.

Actually there are several reasons for this observation. The most probable one is the protonation effect of even the amino acids in the active site. BSA is a very interesting protein, it some time loosely binds to the allosteric site of an enzyme reducing this protonation and many a time removes the steric hindrance that reduces the strength of the bond between the amino acids of the active sites and the substrate at low pH. This has been observed and reported in case of Pepsin (cystein protease) several times.

The pH of a solution can have several effects of the structure and activity of enzymes. For example, pH can have an effect of the state of ionization of acidic or basic amino acids. Acidic amino acids have carboxyl functional groups in their side chains. Basic amino acids have amine functional groups in their side chains. If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to altered protein recognition or an enzyme might become inactive.

Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrate so that either the substrate cannot bind to the active site or it cannot undergo catalysis.

In general enzyme have a pH optimum. However the optimum is not the same for each enzyme.

I agree with that pH affects the activity of enzymes by protonation of diverse groups, both of active site as other site. The latter may produce conformational changes that affect the enzyme kinetics. Concerning it to effect that you observe when you add another protein, for it to act as buffer, it has to be in a considerable amount over the concentration of buffer in your sample. If you measure the pH of the final solution you can easily test this hypothesis. Perhaps it is more likely the hypothesis that it acts as a "chaperone" of your enzyme. Anyway, I agree with Regina in that is necessary more information your system to route the search for the answer to your question. If you give me more information, I gladly try to help you.

Best regards

My colleagues have been studying a crop disease enzyme system for years that behaves similarly to what you’ve mentioned. Their paper (Kemp, et al (http://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI.2004.17.8.888)) looks at the activity of the enzyme at various pHs with and without a protein inhibitor. Interestingly, they observe that the “inhibitor protein” sometimes actually enhances the activity at the non-optimal pHs. We have recently published a computational paper that hypothesizes that for many of these complexes the total net charge on the complex is significant. For instance, you will find, anecdotally, that most enzymes function optimally at pHs near their PI. As in our situation, an enzyme that is slightly negative (as a result of being at higher than optimal pH) might not be able to even bind our slightly negative substrate. However, in the presence of our inhibitor protein (which is still quite positive), the enzyme picks up a little activity. We suspect that the enzyme/inhibitor complex, having a net positive charge, no longer repels the substrate. In conclusion, pH certainly affects the specific chemistry of the key active site side chains, but also the total net charge on the enzyme and any complexes may also be significant. I can send you a copy of our computational article if you’re interested.

Many factors affect enzyme activity like pH, temperature, substrate concentration, active site etc. Enzymes are proteins only by nature, so have direct relation with the pH of the working environment due to side chains of amino acids and other active groups which ultimately decides the stability- overall charge and activity of an enzyme. When you are adding the enzyme with another protein, it may be stabilizing the net charge and thus keeps the enzyme active at sub optimal pH values.

I agree with the answer of Shital Phuse. I think he seems very much right that pH of proteins is determined by the chain of types of AAs (amino acids present) to keep them native state which is essential for its activity. If pH changes its native structure or stage that means protein might be less active. Thus protein in native state might be having maximum activity at optimum pH.

Changing the pH does indeed change the charge on the AA side chains and may change such things as phosphorylation or methylation of the enzyme if such is necessary for its activation. This, in turn, affects the activity as it affects the avidity with which it binds the substrate and the ability of the enzyme to act upon it and/or the rapidity of this process. You mention its binding to another protein, as well. If this protein is a kind of "co-factor" that can alter enzyme activity, changing the pH may also change the ability of the co-factor to bind to or act upon the enzyme.

I think everyone has so far summed it up. The protein-protein interaction affects the physicochemical properties exhibited. Regarding your question about this in literature, I know there are more than plenty of examples but you'll have to be more specific with your enzymes. Different enzymes have different properties, so you will see different outcomes for different combinations of enzymes.

What kind is your recombinant enzyme? You did not mention the bio-specificity or function of your enzyme. I guess it would be protease enzyme.

Protein and a mixture of amino acids have some bufferic action which can maintain the enzymatic activity at its optimal pH..

I think that you are researcher, and pH useful in enzyme activity is like the ideal one condition for you to do best test. That can take account internal and external conditions. Every reaction has it optimum pH, temperature....

I agree with many of the answers Protonation and deprotonation of the amino acid side chain will affect the anzyme activity. If occurs at the active site it will have more pronounce effects to the enzyme substrate interaction. If occurs at the sides other than active site it will pottentially alter the noncovalent interactions within the molecule and thus alter the enzyme conformation. PH of the solution also affect the ionic characteristic of the substrate which in turn affect also the enzyme substrate interaction. Addition of other protein such as BSA or others to the solution may induce various noncovalent interactions with enzyme or substrate which can open steric hindrance of enzyme and increase the activities or induce nonproductive structural conformation and reduce the activities In your case the former seemed to occur such that you observed increase enzyme activity at suboptimal conditions

Very simple interpretation:

All enzymes are protein. Various factor affect function of enzyme including ph, temp or allosteric factor cofactors. Increase or decrease factor effect enzyme function.

Thanks all for your answer. Sorry for late reply but I was traveling.

I think there's no need to go again with the theory about pH and enzyme activity. I guess the title of my question was misleading. My real question was in the description. And then in the comment I made to refine the question.

In any case I want to thanks Daniel King for the paper link. At last I found what I was looking for. Still the interpretation of those results are a little bit different, and a BSA control in those in vitro experiments would have strengthen the results.

For the ones that need the enzyme and protein interactor (I think there was no need since I'm asking for similar effects in literature): the enzyme is a phosphatase and the protein is a kinase. I also saw the same effect with a dead kinase mutant (a little bit less pronounce). And I didn't see the effect when I didn't include a substrate (a phosphopeptide). I didn't include the phosphopeptide bc I wanted to distinguished between real dephosphorilation and a phosphorilation-dephosphorilation cycle between the proteins (the same as when I used a dead kinase). The readout is free phosphate for phosphate activity.

Still, only Daniel King give me some hope that this is not an artifact with the Figure 3 of his link.

Please guys, forget about the in vitro biochemistry theory and give some in vivo explanation for those observations (in case you think those are really true, or if you think I need to make more controls). Most of you think this is completely normal (theory-wise), but where are those papers for in vitro? And whats the in vivo aim?

Thank you!!

BTW, that figure 3 should have error bars or some kind of statistics, right?

Because the differences between inhibitor added or not in 3A and 3C are rather small ...

because in increase or decrease in PH the imbalance between negative and positive charged AA which are exists in the structure of enzyme is occur for example if the PH is acidic then there will be more positive charges remain in the protein that pull out each other and as a result the structure is destroyed.

Agreed with all above reason particular the brotonation and deprotonation what I can say is that adjust your protein or reaction mixture to the enzyme optimal pH but do consider the temprature too

Dear Fernando,

as already answered by several contributors, the apparent pH-dependency of enzyme activity can be related to the ionization of catalytic residues (such as histidine in enzymes with a Ser-His-Asp catalytic triad) and stability of the enzyme. It is important to know that it can reflect a combination of several pH-dependent steps in the overall catalysis process. I give you one example I am familiar with, that of lipases. Before hydrolyzing their substrate, triglycerides, these enzymes have first to change their conformation, bind to the lipid-water interface, and then hydrolyze an ester bond using a catalytic triad. All these steps are pH-dependent and they impact the overall pH-dependent activity assessed by measuring the reaction products. We know that addying a surfactant or proteins can change the pH activity profile, mainly because they interfere with the enzyme binding at the lipid-water interface. When you add an new parameter, a protein in your case, you have to ask yourself how this can affect the various steps of the catalysis process. If there is no specific interaction between your enzyme and this protein, it is possible it affects the enzyme stability or interaction with the substrate.

Article How Gastric Lipase, an Interfacial Enzyme with a Ser-His-Asp...

Thanks Frédéric,

I think a surfactant is different from a protein. A surfactant is very important for lipases where the interface water-oil is key, and a surfactant would change the physiochemical properties of that interface.

Furthermore, I also think a protein is different from a Co-factor or any other allosteric activator. I think what I see is not even consider allosteric regulation.

Nowadays we have in the literature thousands of protein interactions but not that many studied in a biochemical way. So far no protein-protein interaction that change any enzyme activity has been describe to my knowledge ... I wonder why (and the in vivo relevance of such enzyme modification).

pH is in its nature a global feature of the solution, whereas protein activity can be interpreted as the overall sum of local activities in the active sites of the individual enzyme molecules. We have proposed in a study of triglyceride lipases that pH in the closed form of the active site may very well differ from the 'global' pH. many triglyceride lipases are equipped with a 'lid' that can hinder free access to the active site. Our results were published here :

Quantization of pH: Evidence for acidic activity of triglyceride Lipases

By: Poulsen, KR; Snabe, T; Petersen, EI; et al.

BIOCHEMISTRY Volume: 44 Issue: 34 Pages: 11574-11580 Published: AUG 30 2005

I suggest that your added enzyme adds buffer capacity to the solution, and therefore alters the probability for finding protons in or close to the active site of your enzyme. Another possibility is that your added protein binds to the enzyme of interest. In that case the titratable residues on your enzyme, will sense the electrostatic field from added protein, and the pK values of the titratable residues will change - thus effectively changing the pH activity profile of your enzyme. The protein-enzyme interaction you can investigate by attaching two fluorescent labels that is able to exhibit FRET when in close proximity.

I did not read all comments but I beleive in what Christian Rückert said. in addition, you need to see the PH of the AAs in your protein. most likely is base ph to naturlize your enzyme to have final ph 7.5-8.

That is right, most of enzymes are proteins and their activity usually are pH dependent. Acidic and alkali pH values can increase/decrease the activity of an enzyme according to the protein 's Tertiary and Quaternary Structures.

i think that pH have effect on the enzyme substrate reaction or binding

i think that pH have effect on the enzyme substrate reaction or binding

This is not a simple case. Everything depends on what are these two proteins doing - I suspect that You have added second protein for some reason. Enzymnes not necassarily acti separately and this is complex matter. Is Your substrate opr rteaction product interacting with the second protein?

Amended on 17 June 2017

I also surely severely consider the enzyme kinetics; i.e., pH dependence, glycochain dependence, metal dependence, detergent dependence, heat dependence, reducing agent dependence (such as 2-mercaptoethanol), The Fascio effect (please see file; The Fascio effect), etc. Therefore,

enzyme kinetic study using HPLC-photometric or fluorimetric determination is important. The study about the dependences of enzyme kinetic parameters V, Km, Ki, kcat/Km (Amo), Rep, and Cap on such environmental factors especially on glycoside-chain seems to become important in the future (please see file; J Chrom B BIN LIP Km).

In addition to the reasons mentioned above, the pH, modifying the protonation/deprotonation state of amino acids, can influence the role (e. g. acid or basic catalysis) played during the chemical mechanism of enzyme, while the conformational change, due to the different protonation state, can be also related to the binding substrate or product release steps.

Dear Fernando

I think possibly one experiment could answer whether this protein is stabilizing your enzyme or the substrate. I would propose limited proteolysis and western blotting for evaluation, limited proteolysis will be at the pH where your enzyme has low activity but strong effect with your second protein and the experiment should be under 2 conditions without and with your second protein. If you see less degradation by adding your protein then I would say that this proteinincreases the stability of the enzyme. Under in vivo conditions there are proteins that may do this like chaperons, but there are more informations needed about your enzyme like metals in active centers, iron sulfur clusters .... I hope it helps

The title of the question is a bit misleading. A better title could be: How can a second protein change the pH dependence of my enzyme's activity?

Nevertheless, the two questions are related: if you understand why the activity of yout enzymes decreases at low pH, than it will be much easier to assess the mechanism by which the second protein acts. You have at least two main alternatives:

1) Your enzyme is unstable at low pH, and either it denatures or (if it is multimeric) loses its quaternary assembly. To check if the enzyme is denaturing, you can pre-incubate your enzyme at pH 6 for different times, before measuring activity, and see wether the activity dies completely at longer preincubation times. To assess the loss of quaternary structure, you can test the activity at pH 6 using different total concentrations of the enzyme. The specific activity (activity divided E concentration) would remain constant if subunit dissociation is not an issue, but will disproportionately increase with [E] if dissociation is at play. In the end, if your enzyme loses activity because it denatures or dissociates at low pH, then the second protein must be stabilizing the enzyme structure.

2) The enzyme loses activity at low pH because the rate-limiting step of its reaction is affected by the ionization state of some group on the enzyme (or possibly on the substrate). This is the 'generic' explanation most commonly offered by textbooks, but going a little bit beyond the general statement may be hard. You should first undestand whether low pHs change just Vmax, or just Km, or both parameters, and also how these parameters are affected by addition of the second protein. The second protein, for example, might be increasing the affinity of the enzyme for its substrate, or it may be lowering the pKa of some crucial group on the enzyme, or it might be changing the rate-limiting step of the reaction.

Note that in the two cases above the second protein must act by forming a physical complex with the enzyme This complex may be physiologically relevant (if it is known that the two proteins co-localize in vivo) or it may be purely incidental. If the complex is stable enough, it may also be isolated through chromatograpy.or other separative techniques. Note however that there may be other (less likely) explanation for your pH-dependence, such as:

3) At low pH, your substrate may become unstable or undergo an alternative reaction.In this case the second protein may be stabilizing the substrate, but this effect would be relevant only if you use the second protein at concentrations comparable to those of the substrate.

4) At low pH, your enzyme may loose some important cofactor or (if you change the buffer type) it may be inhibited by some component of the buffer system. The second protein may somehow counteract these effects.

Thanks Alessio for your comment and suggestions!

Your first point is really interesting, but according to literature, this enzyme (at least the family) is supposed to be monomeric. There are no studies about this specific enzyme though.

For the second point, if it changes Km or Vmax, would it make sense that it's only change at low pH? Because I don't see the effect at "normal" pH.

I also like your third point, but I'm using 0.4uM of the protein that interacts with my enzyme, and 25uM of substrate. And in any case if the effect would be due to the substrate only, the other protein I'm adding should be interacting/modifying/stabilizing/ the substrate, right?

You are right with the cofactor. Mg+2 and I defy love this point. I will follow this lead that I think is the most interesting!! Thanks!

Since you seem very experience, do you know any paper reporting something similar in vitro? And, if all my data is correct, does it makes sense in vivo that a protein broaden the pH spectrum of an enzyme?

>For the second point, if it changes Km or Vmax, would it make sense that it's only change at low pH? Because I don't see the effect at "normal" pH.

Your second protein MUST be changing one or both (apparent) catalytic parameters at low pH, otherwise it wouldn't activate the enzyme.

>If the effect would be due to the substrate only, the other protein I'm adding should be interacting/modifying/stabilizing/ the substrate, right?

You are right, in scenario 3 the protein should interact quite stably with the substrate. Perhaps there can be other mechanisms but it seems pointless to imagine such mechanisms without knowing the identity of the proteins you are using and the assay you are performing.

>Since you seem very experience, do you know any paper reporting something similar in vitro?

I did a quick search on Google Scholar... Perhaps this fits your case (look in particular at page 237)? http://dx.doi.org/10.1016/S1074-7613(00)80475

Also look at this: doi: 10.1074/jbc.M408965200

>If all my data is correct, does it makes sense in vivo that a protein broaden the pH spectrum of an enzyme?

If your data point to a physical interaction between the two proteins, that is certainly a useful hint. I mean, if your protein binds to the enzyme and assists its function in vitro, something similar may happen in vivo, too. Consider that within a cell the local pH can change significantly and be quite acidic, e.g. in lysosomes or near the surface of membranes.

Aside from protein stability, enzyme activity and substrate binding can also be functions of pH and can indicate key catalytic or binding residues. Activity and/or Km values can change with respect to pH by altering the protonation state of key residues in the active site. The pKa of side chains is influenced by the residues surrounding them.

This is because of changing hydrogen bonding due tom the change of protein. When changing hydrogen bonding, protein cannot maintain their original structure, therefore inactivate. This can also same as in DNA and RNA because they also maintain hydrogen boning A-T, G-C or A-U (RNA).

Do you know if the protein you are using possesses peptidase-like activity that may somehow activate your enzyme even at pH 5? This is a long-shot but may be worth testing with a cocktail of peptidase inhibitors, for example.

To my knowledge it doesn't have peptidase activity.

Why would that increase the activity at low pH?

In my understanding that would mean cleavage of the enzyme and less activity ...

Because some of these recombinant enzymes are synthesized as pro-enzymes with a short peptidic chain (N or C-terminal) that renders them far less active unless they are pre-cleaved by dipeptidyl-peptidases. Some serine proteases are activated in some cases by cathepsin C like enzymes.

Of course, this is only a hypothesis with regards to your question.

Regards

Hi

The other protein is interacting with your enzyme and cause some conformational changes, so your protein has new third structure and the function of proteins is tightly related to their third structure. pH affects third structure and function of protein too. So your enzyme with new structure has function in different pH in comparison to enzyme alone. Your protein is enzyme so be sure that other protein has changed the structure of the active site.

Each enzyme has its own PH at which it is highly active that called optimum pH for that particular protein. The two enzymes acid phosphatase and alkaline phapsotase catalyses same reaction but have different PH for maximum activity and location and hence their name acid and alkaine phosphatse requiring particular PH for max. activity. As their pH of particular enzymatic activity get changed it will get start to denature the protein (enzyme), and automatically the activity will decrease.

You may use a little help from computational biologist, protein molecules are the same as other molecules, which has it's own PH. When you mix two different molecules into one solution, the interaction between two proteins will effect the hydrolization state of interacting side chains, if the interaction is happending in the active site, the activity may be changed. when you lower or increase the PH of the whole system, the hydrolyzation state of active site may change again....

Enzymatic reactions depend closely on the interaction between the enzyme and its substrate. Enzymes present various structural conformations which are pH dependent. The optimal activity being obtained when the conformation is ideal for substrate fixation on the enzyme's active site. Other proteins in the medium may enhance this activity by stabilising the enzyme at its ideal conformation although at a pH different from the optimal pH.

I had the same phenomenon with an enzyme I am studying. I found that two cofactors were required for full activity, and that the presence of both dramatically broadened the pH profile. In my case the activator could be any polybasic protein in vitro, and BSA did reasonably well (the region of BSA that binds fatty acids contains a number of basic residues - using fraction V BSA that has been delipidated exposed these residues).

The activator you have found suggests, as others have noted, that it is stabilizing the enzyme active site, or altering the pKa of the active site residue (shielding, conformational change, water binding or exclusion, etc, etc.) It may even be causing your enzyme to oligomerize or detach from inactive oligomers - too many possibilities to be helpful.

Here if the link to my initial paper, in case you find it helpful. The change in the pH profile is in an AER paper - I'll try to make sure it's under my profile if you need it.

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.404855

Most of answers are enthusiastically educational and well understood. They are appreciated. It is all right if no other factor involved.

May I ask about some other factors which may not follow the same pH mechanism . So what mechanism occurs with t those temperature-dependent enzymes which may be only pH-dependent.

Proteins must be very tightly regulated in order to maintain the proper equilibriums in their environment. Everything is pushed and pulled by electrons and the lack of electrons at a given site. When an enzyme is sensitive to a particular pH, there is usually an amino acid residue which has a protonatable site present in a very critical site. The status of the site whether it is protonated or negatively charged, is governed by the electronics of the neighboring moieties which join together in a charge distribution causing the site to protonate at a particular pH. The status of these charges and the surrounding media pH can cause a protein to totally change its tertiary structure....folding up or opening out which can sequester or hide other key sites or conversely make them available to the binding of other factors. It is an exquisitely sensitive dance that balances on basic structure to keep all in balance. I tend to think of enzymes as if they were individual organisms because they are so unique and complex.

pH can have an effect of the state of ionization of acidic or basic amino acids. Therefore, if the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the protein structure can be altered. Accordingly, this can lead to a reduction of protein activity or an enzyme can assume a non catalytic conformation.

pH can affect protein conformation and thereby activity. pH alters the state of ionization of acidic or basic amino acids. If the state of ionization of amino acids in a protein is altered then the ionic bonds that help to determine the protein structure can be altered. This would affect the active site or ligand interactions and change activity. Enzymes do have an optimum pH. Binding with proteins and ligands would affect the conformation and activity. To get more information from the book on Enzymes by Dixon and Web or even the chapter of enzymes in a standard Biochemistry text like Leninger.

Watch out for the titratable side chain aa's.

The enzyme and protein are bonded with hydrogen boning. If pH change, the original hydrogen bonding differ, indicating original structure cannot maintain. This result in enzyme activity change. This is the reason enzyme and protein structure is pH dependance..

Read Dixon's Book: Enzymes!

There is not an universl answer for all the enzimes. Certain enzime are active in a broad range of pH, while other not. of course extreme pH significativelly alter the ionization state of the charge residues and their functionality and ability to interact and bind possible cofactors.

for example: if you have a zinc metalloprotease that bind zinc by histidine is most probable that its activity will be strongly reduced at pH

pH affects charged amino acids (their ionization status). pH also can affect structure of the proteins. so your enzyme might change structure (hidden vs open state), as well if your enzyme is co-factor dependent (metal: Mg, Zn etc) ability to bind co-factors and be activated would be affected.

to understand the effect of pH in protein (Enzyme) , referred to isoelectric point (PI of Amino acid). The ionize form of protein may decrease or increase the activity of enzyme.

Like other bioactive proteins, enzyme-proteins have biological specific functions which are related with their primary structures and specific foldings.

pH has an effect on amino acid protonation state and therefore to enzyme conformation and activity. it is mainly histidine that is responsible for this protonation change as its imidazole side chain has a pKa of approximately 6.0. The result is that, around physiologically relevant pH values, relatively small shifts in pH will change its average charge.

What the hell does the enzyme work on? Don't you think that is important information for this question? Many enzymes operate on general acid- or base-catalysis, and so the ionic state of the relevant amino-acid side chains at the active site is crucial to the activity. (the pH dependence suggests a histidine residue). On the other hand, while pH5.5-6.0 is not usually low enough to cause partial or complete unfolding, the activity-stabilizing effect of your unnamed protein does suggest that it might be inhibiting the unfolding by compkexation. Reading Dixon, or any good biochemistry textbook, probably would have answered your question.

Arnold, thanks for you polite comment.

As I said in the title, please read previous comment first.

The biological meaning of pH activity dependence of enzymes and their interactions with different proteins and cofactors is related to their function and compartamentalization in different cell organelles, tissues and organs.

Fernando:

Your bi-function enzyme with optimum at pH 5.5 and 6.5 mixed with other protein, you found that it was interacting with your enzyme and expands the pH spectrum at which the enzyme is fully active.

I ask:

What is the protein you mixed, since it was not BSA? Did it happen with several other proteines.

My guess is that your enzyme interact either combine or associate with the protein which acted as a buffer.

The enzymatic activity depends directly of pH range, because the lateral chains of active site can be protonated or not. The other added enzyme which increase the activity of the enzyme of interest may be due to a substrate present is catalyzed in the reaction and their product can increase the final pH in the reaction. Did you check the pH after to add the second enzyme?

Enzyme activity is dependent of conformation of the molecule. Special importance is the conformation of the active syte. pH is influencing both.

Protein-protein interactions may have simillar or special effects on the conformation of the active syte or the pH dependent conformation changes.

Experimental results may explain the phenomenon. Especially in case of protein effect of the activity pH dependence.

The pH of a solution depend on the ionization state of acidic or basic amino acids. Acidic amino acids have carboxyl so the protein has an effect of the activity pH dependence.

Hi Fernando,

The effect of pH on enzyme activity is complex. Some enzymes are quite stable with reference to enzyme activity in the high pH range, some are more stable in the lower pH range and some are quite stable in the broader pH range. Generally many enzymes show a bell-shaped pH-activity profile with a pH optima. Loss of enzyme activity at the extreme pH conditions can be ascribed to protein denaturation (acid denaturation or alkaline denaturation) where the enzyme's 3-D structure is lost. However, within a certain pH range, loss in the enzyme activity may be attributed to protonation or deprotonation of ionizable catalytic groups present in the active site of the enzyme.  

Your observation of increased enzyme activity in the lower pH range in the presence of another protein simply reflects enzyme stabilization by another protein. 

Hi Saad,

Protonation or deprotonation  may take place at different  parts of the protein molecule. Events like this - as a consequence -  may change the coformation of the active syte.

Hi Erdei,

As I mentioned in my reply that pH effect on the enzyme activity is complex. There are several theories. According to acid-base theory of enzyme catalysis, bell-shaped pH-activity profile is made of two titration curves of the ionizable catalytic groups. For example in lysozyme-catalysed hydrolysis of chitin (pH optima is ~5.0), decrease in the enzyme activity towards acidic range can be explained on the basis of protonation of Asp52 (catalytic residue, which is required in deprotonated form to stabilize the carbonium ion intermediate) while decrease in the enzyme activity towards alkaline range can be attributed to the deprotonation of Glu35 (catalytic residue which is required in protonated form to act as proton donor to start the cleavage of glycosidic bond).  That is why I said that loss of enzyme activity might be attributed to protonation/deprotonation of the ionizable groups present in the active site. Getting a deformed active site conformation requires major protonation or deprotonation reactions of a large number of ionizable groups.

Hi Saad,

Your explanation concerning the effect of pH on the two residues is clear, and ok.  What I was stressing on pH has influence on all ionisable groupsof the protein molecule: This may induce  conformation changes of the active syte.  This way influencing catalytic activity.

Hi Erdei,

I have already mentioned this in my previous reply  (Please see last three lines).

Hí Saad, It is ok

Dear Fernando,

I assume that your negative control (BSA) did not change the pH profile? Instead at the most I assume BSA might have stabilized/increased the overall activity? What happened?

With the addition of the protein in question you are seeing a drastic shift on your pH profile in terms of enzyme activity. This means there are titratable residues that are changing the active site ionization status. In other words, upon addition of your protein "Q" you are some how  titrating your active site residues of protein "P" and making it active even at the so called pH 5? Did you measure the pH of the media before and after the addition of the protein Q? What concentration of protein Q did you Use?

Are you changing your overall protein fold? Did you perform any spectroscopy?

Physiological Significance: What kind of protein is it? that you added?

Remember: Intracellularly lysosomal compartment is very acidic (~5) compared to cytosolic pH (~7). Intracellular (lysosomal) pH's I would assume are controlled/managed by proteins and degradative products of DNA, RNA etc? as opposed to cytosolic pH (controlled mostly by phosphate buffer)

Whatever it is this is quite interesting. If you heat denature the added protein "Q" do you see the same effect?

You know there are isozymes/forms that have similar folds but differ slightly in the active site and neighbouring residues. Interesting.

Venk

I think the similar way as replied by Venk.

Most of the answer can be summarized as follows:

A) pH has two separate effects of enzymes, that may or may not be easy to dissect.

- As elaborated by Saad, large pH changes affect protein stability. Ligand binding (and proteins can be very specific ligands) can add stability to the complex, and such is the basis of the thermal shift assay.

-As described by Christian, subtle changes may change the ionizations state of the active or regulatory sites and modify the kinetic response of proteins (affinities, catalytic constants,  specificity constants, and allosteric regulation).

B) In addition, as mentioned by Shital,   protein-protein interactions may ( and usually do) change conformation of the proteins in the complex, relative to the free proteins. As a result of the exposure or burial of important groups in the enzyme, a conformational change may shift selected pKa values leading to a modified enzyme's response to pH.

THEN:

You need to test is your enzyme is stable in the whole pH range, by incubation of samples at several pH values and determination of the remaining activity in aliquots taken at increasing incubation and assayed at a constant pH, in an optimum or standard assay.

You need to see if the low activity at pH 5 is due to a reduced binding (kCAT/KM vs. pH assay values) for the substrate(s) or to a change in kCAT (or both). A reduced affinity for the substrate may result is an assay being saturating at high pH and well below saturation at low pH. That may requires a series of saturation curves (activity vs. substrate concentration) at several pH values in the range of interest. If your enzyme has more than one substrate this is hard work.

The test proposed by Kallidaikurichi is also important, because you need to see is the effect of the added protein is specific or not.