Let's be clear about the experiment you are doing.

To measure Vmax, you measure the initial rate of the reaction at a range of different substrate concentrations, plot a graph of initial rate versus substrate concentration, and fit the data to the Michaelis-Menten equation, observing the requirement of the Michaelis-Menten approximation that the enzyme active site concentration is always much lower then the substrate concentration. Vmax is the initial rate where this curve reaches an asymptote at saturating substrate concentration.

It sounds to me like you may be doing something else: adding more substrate to a reaction after the rate of product formation has leveled off (i.e. the rate is zero). In that case, the resumption of product formation after adding more substrate is expected because the substrate was previously depleted. It is possible that it may even have reached zero. Therefore, the enzyme was not saturated with substrate at that point.

Adam, firstly, let me thank you for timely responses to the questions I post on research gate. You have been very helpful. Secondly, I think the way I asked the question led you to misunderstand my experiment. I am in fact doing individual reactions at specific concentrations of substrate while keeping enzyme concentration constant as you stated in your reply. I then calculate the specific activity of the enzyme at each substrate concentration using the linear region (i.e. no asymptote) and plot a graph of the substrate concentration versus the specific activity. When I make this plot, I see the rectangular hyperbola characterstic of enzymes following the MM kinetics model, which includes the region where the kinetics appears to be reaching Vmax. But when data points from higher substrate concentrations are added to the plot, then instead of reaching Vmax as hinted, I see an increase again. I hope this clarifies things. 

Hi there,

You may have 2 classes of catalytic sites : one with low Km and the other with high Km. The 2 Km being very different. Do you eventually manage to observe saturation at very high substrate concentration?

Dominique, I have done some more assays and I think I am approaching "another saturation point" at higher concentrations; I do need to try some higher concentrations to see if the graph plateaus out. Also, the "first" and "second" parts of the graph with a "plateau" between them look similar. Do you mean that there could be two active sites? 

Is your enzyme preparation pure enough to be sure that there are not 2 or more different enzymes performing the same reaction, each with its own Km?

Another possibility to consider is that the substrate is unstable and spontaneously forms product, this effect only being observable at high substrate concentrations. The spontaneous rate can be subtracted from the catalyzed rate by making a blank measurement without the enzyme.

I once had a situation in which a coupling enzyme was used to detect the product of an enzyme reaction, but the coupling enzyme was able to consume the substrate of the primary enzyme at a low rate. Is this a possibility in your system?

Adam, to your first question: the enzyme is expressed with a 6x His-tag and then purified with Nickel resin. I then collect the purest fractions following imidazole elution. Of course, this does not ensure 100% purity, but I have performed assays with other substrates that can also be catalyzed by the enzyme. The substrates share the same functionality (that is catalyzed by the enzyme) albeit structural differences are present between them and the substrate being discussed on this page. Since I have not noted odd kinetic curves with other substrates from the same batch of purified enzyme, the fact that another enzyme could also be catalyzing the same reaction is dubious. 

To your second comment: I did have a similar thought and I ran some assays to test the background activity by including all the reagents and the substrate and excluding the enzyme. I did this reaction at the high concentrations showing aberrant activity relative to the MM curves I normally get with enzyme when I have extracted kinetic data from its activity on other substrates. However, I did not see any background activity so it looks like the activity increases can be attributed solely to enzymatic activity. 

So far, I have not added any coupling enzymes into the reaction. 

Is this a homooligomeric enzyme, allowing the possibility that there may be multiple interacting binding sites?

Is it possible that there is an allosteric binding site for the substrate? Do you have a way of measuring the binding stoichiometry of this substrate?

Adam, I also thought about that. Perhaps it is a dimer or oligomer of sorts...but since I do not see this kind of kinetics with the native substrate I wasn't sure what to make of that hypothesis. However, the substrate in discussion is not the native substrate and it is catalyzed at a lower rate. This may explain why we can detect these nuances in its kinetics graph as compared to the native substrate. I have not yet thought of how to measure binding stoichiometry so any suggestions that you may have about that are welcome. There is no a crystal structure of the full enzyme as of yet. 

It is challenging or imposssible to directly measure the substrate binding stoichiometry if the reaction proceeds when you add the substrate. In some cases, there is a cosubstrate or cofactor that is needed for catalysis. In those cases, leaving out the cosubstrate or cofactor can let you measure the binding stoichiometry. If you have a competitive inhibitor, you can measure the binding stoichiometry of that as a surrogate. There are several ways of doing it. Isothermal titration calorimetry and equilibrium dialysis are a few examples.

I completely agree with the last comments from Adam as equilibrium dialysis usually works well and it is easier than titration calorimetry.

Is it an enzyme catalysing redox reaction? In that case, protein film voltammetry may help.

The effet you describe is seen with true lipases, is your enzyme a lipase?

With substrate concentrations below the solubility limit you get MM kinetics which can reach vmax depending on KM and substrate solubility. Once you reach the solubility limit, the substrate will form a second face (droplets...) and you will get a "new" MM curve with a higher vmax. Do your second MM curve start at the solubility limit of your substrate? The phenomen is called interface activation.

Another idea that occurs to me is that the highest substrate concentrations may alter the Vmax of the enzyme by changing the environment, such as shifting the pH and/or the ionic strength of the buffer.

Anders, my enzyme is not a lipase. As far as the solubility limit, the substrate I am using is very soluble in aqueous environment and I have not reached the solubility limit with the concentrations that I have tested so far. Adam, that is possible. I need to test the pH the next time I do the assay. Thank you all for your help. 

Some enzymes change their oligomerisation state and hence activity depending on [S], so called morpheins (proteins that morph). PBGS in haeme synthesis is an example.

Surely hydrophobic substrate can not be dissolved easily.   Then, enzyme kinetics are measured at the concentration around the Km values. 

I have recently answered with respect to hydrophobic membrane glycoprotein enzyme from Dr. Sammy Pontrelli (University of California, Los Angeles (UCLA), Department of Chemical and Biomolecular Engineering, Los Angeles, CA, USA).

I have used expensive and hydrophobic substrate BAQ at low concentrations.

I always am recommending the reliable and sensive HPLC-photometric enzyme assay (please see file; J Chrom B BIN LIP Km).   HPLC method requires the substrate in very small amount; i.e., only 0.1 mL of reaction mixture is required to perform the enzymatic reaction.   By the way, Sigma has previously stopped selling our important substrate of BAQ (biotinyl-6-aminoquinoline), but HPLC method can be performed to publish the above article due to minimum use of substrate (measurement of initial velocity at low substrate concentrations). 

We have previously developed an HPLC-photometric-carboxylase assay in order to get rid of the use of dangerous radio-isotope (please see file; 4-kinds of carboxylase). 

Further, such a characteristics is observable in hydrophobic membrane glycoprotein enzymes such as thiol-type rat brain and rat kidney biotinidases.   Then, I have diluted the homogenate, soluble, and membrane fractions containing enzymes by 0.1 M sodium-phosphate buffer (pH 6.8; with 1 mM EDTA) in series and biotinidase activity have been determined (10 mM 2-mercaptoethanol (2-ME) has been added immediately before the start of the reaction).   The infinitely-diluted enzyme activity has been estimated graphically and expressed as Vmax or V.   I am now considering that the active center of the membrane glycoprotein enzyme seems to be hidden by hydrophobic molecules of membranes.   It is noteworthy that such an assay with dilution can uniquely performable only by using sensitive HPLC-photometric enzyme-assay method.   Further, safe and successful dilution can be easily be performed by using sensitive, quick, and reliable HPLC-protein assay method (please see file; HPLC-SEC-protein determination method).