The first question is whether you measured the initial velocity at each substrate concentration. Since you said that you made a single product measurement at 15 minutes, it is probable that some of the reactions, especially the ones with the lowest substrate concentrations, had slowed down by that time so that the initial rate was underestimated. It is probably for this reason that your double reciprocal plot is not linear. There could be other reasons, but this is a good place to start.

To make sure you measure initial velocities, you should take several time points at each substrate concentration. Plot the product concentration versus the reaction time and measure the slope at the beginning where it is steepest. If the reaction goes too fast to make the slope measurement, repeat with a lower enzyme concentration.

which kit  that used ?

The first question is whether you measured the initial velocity at each substrate concentration. Since you said that you made a single product measurement at 15 minutes, it is probable that some of the reactions, especially the ones with the lowest substrate concentrations, had slowed down by that time so that the initial rate was underestimated. It is probably for this reason that your double reciprocal plot is not linear. There could be other reasons, but this is a good place to start.

To make sure you measure initial velocities, you should take several time points at each substrate concentration. Plot the product concentration versus the reaction time and measure the slope at the beginning where it is steepest. If the reaction goes too fast to make the slope measurement, repeat with a lower enzyme concentration.

Thanks Adam B Shapiro so much. I did not measured the initial velocity at each substrate concentration. So I will try this way.

Additionally, I'd like to make clearly that my enzyme only make a single product because I already comfirmed by HPLC.

Once again, thank you for your advice.

you need, first, to optimize your reaction using a minimum required amount of the enzyme and substrate, second, to take time points of substrate breakdown within the window of linear initial reaction rates. 

Hi  Tien,

Firstly, you need to optimize your reaction system, amount of enzyme required to hydrolyze the substrate and other reaction conditions.

The graph showed that the minimum concentration that you used is quite high, you can take the starting concentration much lower than previously used to make the parabolic curve.

With parallel increase in the concentration  the curve will be parabolic or sigmoidal based upon the nature of your enzyme. 

After saturation the a constant value of activity will give you the right Vmax.

I hope it will help.

Best luck

Ashok

@ Hamad: Thank you so much

@ Ashok K. Nadda: Thank you for your advice. I also found the problem as you mentioned. When I used low concentraion of subtrate, the curve was fine, but when I tried with high concentration of subtrate ( over 70 mM), my enzyme did not follow Michaelis - Menten equation. So, when I drawn the graph, I got rid of the data that did not follow Michaelis - Menten equation. 

This is the first time I try enzyme kinetic, so your advices are very useful.

Thank you so much.

Best 

Tien

Primerely, you need to optimize your reaction with respect to enzyme cocnetration

The given graph illustrates minimum enzyme concentration used for reaction is quite high, and so you should take lower amount of enzyme to draw a graph with accuracy.

After saturation, a constant value of enzyme activity will suggests right Vmax.

I hope it will help.

If you are using pure enzyme, you should try a simple reaction using a moderate fixed [S]  and follow the reaction over several time points to see if the formation of [P] is linear over this time course.   Secondly, you should measure the rate of the reaction over several time points using fixed [S] and increasing [E] again to see if the reaction is linear and over how many time points.

Once you have done these basic tests, you should set up a range of [S] starting from very low to quite high depending on the sensitivity of your assay and detection (of your product) procedures.   After bringing the [S] to equilibrium at the desired temperature, you should add an aliquot of E and start the timer, and take aliquots of your reaction mixture at time points, stop the reaction immediately and measure the [P].   Plot either the [S] remaining (less accurate) or [P] formed versus time for each concentration of S used in your measurements, calculate the slope of the reaction  and from that you can calculate V.   Once you have got the V values for all [S] used, you can use ENZFitter software to calculate the Vmax and also the Km for your S, at the pH you are performing the assays.

 Vmax divided by the [E] will give you the Specific Activity.

I hope this is helpful.

Hello,

As a point of interest, the double reciprocal plot (Lineweaver-Burk) is an outdated, biased method of measuring enzyme kinetics and determining Km and Vmax. It has been shown to be consistently unreliable and should not be used anymore. See this review for an explanation :

http://onlinelibrary.wiley.com/doi/10.1016/S0307-4412(96)00089-1/pdf

"The double reciprocal method is consistently the worst method of estimating Km and Vmax values"

It is generally better to do nonlinear fit of your data with the least-squares method. Here is a guide on how to achieve it with Excel, it's very easy :  https://web.archive.org/web/20170709004809/http://src.sfasu.edu/~avk/BTC560/HOW%20TO.htm

You only need to install the Solver add-in (which will then show up in the "Data" tab of recent Excel versions) and follow these steps.

Some statistics software such as Graphpad Prism can do it even easier.

Good Luck,

Ivan Weinsanto