Lineweaver-Burke and some peculiar results

  • Thread starter nobahar
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In summary, yggdrasil is trying to figure out what might be wrong with his experiment after performing a fumurate/malate reaction, one with succinate as an inhibitor and one without succinate. After calculating the rates and plotting a lineweaver-burke plot, he found that the final data point for the + inhibitor case shows a huge difference from the other data. He is not sure if human errors were involved in the experiment, but he feels a bit more confidet now.
  • #1
nobahar
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Hey guys!
Really need some help...
Not sure if anyone is familiar with this, but I performed a fumurate/malate reaction, one with and one without succinate as an inhibitor. However, after performing the calculations and then attempting to plot a lineweaver-burke plot i got the following results!
substrate 0.01 0.006 0.004 0.003 0.002 0.001
1/substrate 100 167 250 333 500 1000
1/velocity + inhibitor 5.34 11.78 16.91 26.21 36.13 45.69
1/v no inhibitor 3.10 3.268 3.356 3.876 4.098 6.329

See my surprise! the final one shows a huge difference! I realize taking the reciprocal poses exaggerates differences... but do these results seem plausible? Should they be that high numbers for 1/v+inhibitor?
I would really appreciate any help, I'm not sure where to go from here... I could claim human errors in the experiment, but I don't know if these are wrong...
Thanks.
 
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  • #2
Can you explain what issues you are having in a little more detail? What "final one" are you referring to? Are you commenting that the last data point for the + inhibitor case seems to differ from the other data? In this case, the 45.69 1/v measurement represents a very small rate that is very susceptible to error.
 
  • #3
sorry, I've been mulling over it for ages and I forget to explain it in enough detail...
yeah, basically the 1/v with inhibitor seems really different from without, the 1/v value; it seems way too high at the lower concentrations (which would be the 1000 end, as you know). 45.69 with inhibitor compared to 6.34 without.
 
  • #4
The data don't look so odd to me. Inhibitors are supposed to decrease the rates of chemical reactions. Is there any reason why you would think that the inhibitor would not be an effective inhibitor? What concentration were you using compared to its binding constant and compared to the concentration of enzyme?

If you plot out the data on a linear plot (reaction velocity v. [substrate]), it looks like the - inhibitor data shows a typical hyperbolic curve with a Km around 0.001 (in whatever units your substrate concentration is measured). The inhibitor, assuming it is a competitive inhibitor, looks like it would have a Km of around 0.01. A change in the apparent Km of an enzyme of 10 fold seems reasonable to me.
 
  • #5
Thanks yggdrasil, I feel a bit more confidet now. I played around with the numbers a bit and found that a small chage in absorbance readings throws it right up or down. I'm hopeful that the readings are okay, as if I had made a slightly different one, the plot comes out fine...
Thanks again!
 

1. What is the Lineweaver-Burke plot and how is it used in scientific research?

The Lineweaver-Burke plot is a graphical representation of enzyme kinetics data, which is used to determine the kinetic parameters of an enzyme. It helps to visualize the relationship between the substrate concentration and the reaction rate, allowing for the determination of the Vmax and Km values of an enzyme.

2. What are some of the peculiar results that have been found using the Lineweaver-Burke plot?

Some peculiar results that have been found using the Lineweaver-Burke plot include non-linear plots, which indicate that the enzyme is not following traditional Michaelis-Menten kinetics. This could be due to the presence of multiple enzyme forms, enzyme inhibition, or enzyme cooperativity.

3. How accurate and reliable is the Lineweaver-Burke plot in determining enzyme kinetics?

The Lineweaver-Burke plot has been widely used in scientific research and has proven to be a useful tool for determining enzyme kinetics. However, it is not without its limitations and must be interpreted carefully, as experimental errors and other factors can affect the accuracy and reliability of the results.

4. Can the Lineweaver-Burke plot be used for all types of enzymes?

No, the Lineweaver-Burke plot is not suitable for all types of enzymes. It is most commonly used for enzymes that follow Michaelis-Menten kinetics, which is a simple model that assumes a single substrate and a single enzyme form. For enzymes that do not follow this model, alternative methods may be necessary.

5. How has the Lineweaver-Burke plot contributed to our understanding of enzyme kinetics?

The Lineweaver-Burke plot has been a valuable tool in enzyme kinetics research, allowing scientists to determine the kinetic parameters of enzymes and understand their mechanisms of action. It has also led to the discovery of new enzyme forms and helped to identify inhibitors and other factors that can affect enzyme activity.

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