Enzyme hydrolysis reaction are carried out using alpha chymotrypsin as an enzyme. Substrate concentration dependent hydrolysis rate are carried out in a miceller medium.
Hi there,
The answer is no if your enzyme is indeed michaelian. If it is there is definitely something wrong with your data. Are you absolutely sure about the substrate concentrations used for the assays because if they are overestimated (especially for low concentrations) points will be shifted to the left modifying slope of the curve and therefore intercepts with axis will be modified.
Hi there, I looked at the file you attached. It looks like you are using the equation of the line to determine the y- intercept which should be equal to 1/vmax. What you can do is extend the line so that it crosses the y-axis, that should be actual y-intercept. You can also check that this is correct by making a separate Michaelis-Menten plot using Prism6 software, divide the 1 over the value of Vmax you get and see if it is a similar value on the Lineweaver -Burk plot. If the Vmax value is still negative then you need to check your assay
hope this helps
I agree with both the above. It is probably your assay methodology. (Although- I have had experience with an incredibly slow enzyme reaction which was virtually impossible to analyse) I think you should have a look at Segal's book (Enzyme Kinetics).
Thank you all for your suggestions
I have measured the substrate/product concentration using absorption spectroscopy. The rate of hydrolysis reactions for a particular substrate has been obtained by linear fitting of [product] vs time curve. Each measurement has been repeated for 3 times. The enzyme essay data reproduce an intercept of 0.35 (sec) with slope 500 (μM.sec) when the hydrolysis reaction are carried out in a buffer (pH~7) medium. However the intercept is always (-)ve in micellar medium. One thing is that the enzyme structure does not remain same in micellar medium as it in buffer. Same type of (-)ve intercept obtained when the essay has been carried out in guanidine hydrochloride solution. So my question is, are there any relation between –ve intercept and enzyme structure?
Dear Animesh,
your result might have come due to the purely technical inconsistency, which in effect has nothing to do with any sensible fundamental background...
Why wouldn't you try using other approaches, like Hanes/Eadie-Hofstee?
Below is just what Keith Wilson and John Walker conclude in their comprehensive treatise "Principles and Techniques of Biochemistry and Molecular Biology", 2010:
"... It can be seen that the Lineweaver–Burk equation gives an unequal distribution of points and greater emphasis to the points at low substrate concentration that are subject to the greatest experimental error whilst the Eadie–Hofstee equation and the Hanes equation give a better distribution of points. In the case of the Hanes plot, greater emphasis is placed on the experimental data at higher substrate concentrations and on balance it is the statistically preferred plot. In spite of their widespread use, these linear transformations of enzyme kinetic data are subject to error. Specifically, they assume that the scatter of points around the line follows a Gaussian distribution and that the standard deviation of each point is the same. In practice this is rarely true. With the advent of widely available non-linear regression software packages such as DynaFit (www.biokin.com) and BRENDA (www.brenda-enzymes.info), there are now strong arguments for their preferential use in cases where accurate kinetic data are required."
... Only after the properly throughout and careful data analysis would it be possible to draw definite conclusions about the mechanism you are attacking...
Respectfully yours,
Evgeni B. Starikov
Dear Animesh,
You will find an explanation in our work obtainable at researchgate, or get it from arxiv.
Best wishes,
Kamal Bhattacharyya
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