Lineweaver-Burke and some peculiar results

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The discussion revolves around unexpected results from a Lineweaver-Burke plot analysis of a fumarate/malate reaction with and without succinate as an inhibitor. The user observed a significant difference in the 1/velocity values, particularly with the inhibitor, raising concerns about the plausibility of the results. Participants noted that high 1/v values at lower substrate concentrations can indicate susceptibility to error and suggested that the inhibitor's effectiveness should be considered. The data appeared consistent with expected behavior for a competitive inhibitor, showing a reasonable change in apparent Km values. The user expressed increased confidence after considering the impact of small changes in absorbance readings on the results.
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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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.
 
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.
 
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.
 
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!
 

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