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All Answers (12)

3rd Mar, 2013

Christopher Gaughan

Zymera, Inc

Thanks Toufic, I think intuitively it should work, but the Michaelis Menten equation was derived under a specific set of assumptions, where [E]>>[S]. I was wondering if there was a convenient way of getting such parameters of v vs [E] rather than v vs [S]

3rd Mar, 2013

Rodrigo Gonzalo Torres Sáez

Instituto Colombiano del Petroleo

I recommend you Using of the excellent book "Biochemical Calculations" from I. Segel. This book will give you a great approach for your research on Enzyme kinetics.

3rd Mar, 2013

Ted M. Lakowski

University of Manitoba

I strongly suggest that you use non-linear least squares fitting. Programs such as Sigma plot and graph pad have the Michaelis Menten equation in the software you simply select the equation with the nonlinear regression wizard and the Km and Vmax will be derived for you without transformation of the data. [S]>>>[E] is a requirement for invoking MM kinetics but you also need to show that there is a linear increase in product formation with respect to time over the period you measure kinetics if you are continuous measurement of your initial rate. However this is even more important if you are only measuring a single time point for initial rate. You will also need to demonstrate that less than 10% of S is converted into P as MM kinetics also assumes negligible formation of product. Please consult the many books by R. A. Copeland he will tell you everything you need to know in a concise and easy to understand format. Segel's book is for more complex reactions than S+ E---> ES ---> E+ P. Hope this helps

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3rd Mar, 2013

Boopathi Subramani

Academia Sinica

There is a simple method available for non-linear least square fitting without using any special programs. You just need to use Excel spreadsheet and activate the 'Solver' in Add-ins. You can refer the following article for more details.

Gerdi Kemmer & Sandro Keller

Nonlinear least-squares data fitting in Excel spreadsheets.

Nature Protocols 5, 267 - 281 (2010)

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4th Apr, 2013

Luke Alderwick

University of Birmingham

If you are performing a reaction where [S]>>[E], then you must assume that your enzyme is still following "Zero Order Reaction Kinetics". Basically, what this means is, as you increase your enzyme concentration, your observed/measured rate of reaction will increase proportionally in a linear fashion. You should not really see a "curve", in the sense of a MM hyperbolic isotherm, because essentially, you will always saturate out the total number of substrate binding site because, by definition, [S]>>{E]. What you should see is a straight line following y=mx+c. It may drift off as you reach higher [E], but this might be due to experimental set up, such as limitations of measurement of your reaction (i.e., analysis moving out of the linear range on your detector). Only when you titrate icreasing [S] against a fixed [E] will you observe classical MM curve (assuming your enyme follows "standard MM kinetics".

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4th Apr, 2013

Tomáš Hluska

The Czech Academy of Sciences

@Christopher Gaughan - if they assumed [E] >> [S], than you would not see the saturation of enzyme with the substrate (i.e. the graph would be linear).

Theoretically, you can plot 1/v vs. 1/[E], but you must realize, that the "Km" you will get from such graph is not Michaelis-Menten constant of substrate, but it is concentration of enzyme at which the velocity is half of maximal velocity!

4th Apr, 2013

Christopher Gaughan

Zymera, Inc

Thanks for all the answers everybody. I did indeed do this experiment and obtained a the classic 'curvilinear' plot one sees with MM. Since the reaction product is fluorescent, I was able to tract the reaction and plot [E] (x-axis) vs Relative Light Units (RLU's). I believe Luke above got it most right, however: when [E] > Km (usually 100 times Km, so you get 0.99% kcat, which is close enough considering experimental error).

I suggest you read chapter 2 (pg 18 to 99) of

Segel, HI. Enzyme Kinetics, Willey, 1993.

best wishes

Rogelio

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5th May, 2013

Paul C Engel

University College Dublin

If I understand your question correctly, the answer is really 'No'. You should expect that if [S] is constant and you vary [E] the rate you measure should be strictly proportional to [E]. It is an important thing to test, but it is an underlying assumption in most of what we do. If that is not what you see when you plot it, it could means several things:

1. You've got into a range where the reaction is so fast that you can't accurately estimate the initial rate;

2. The enzyme preparation contains an inhibitor or na activator which is getting diluted or concentrated with the enzyme itself;

3. If it is an oligomeric enzyme, perhaps it is dissociating at low [E] and giving anomalously low rates.

Depends exactly what you are seeing, but, no, you can't get Km etc. from the plot you envisage.

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11th Nov, 2013

Marcia Moss

BioZyme, Inc. Apex, NC

1/v vs 1/e could tell you as Paul suggested that you have a multimedia enzyme, ie for,example a monomer vs dimer equilibrium, and one form could be more active than the other. So this type of experiment, where the enzyme concentration is varied and kinetics are followed is a standard way to look at multimeric enzymes.

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