Enzyme kinetics Km Vmax Alcoholdehydrogenase

[Almeisan]

So in the lab I have measured my first enzyme reaction rates to determine the Km and Vmax of ADH. We are using yeast ADH.

We are measuring it using DCIP coupled to NADH production using PMS. So NADH does not accumulate and we measure tha lowering in DCIP concentration using spectroscopy.

Ethanol is used as a substrate.

I made a somewhat nice looking lineweaver burk plot but that gives me strange values. I get 2400 umol as a Km value. And my Vmax of 10.4 is lower than the V I measured at the highest concentration.

We are using 0.0075 mg/ml of enzyme. Highest ethanol concentration is 0.0675 M. I have 12 data points and the highest ones haven't reached Vmax yet as there is a 6% increase in reaction rate between the second highest and the highest data point.

More puzzling, that second highest reaction rate is at a concentration 23x higher than my Km. But Km is supposed to be half the substrate concentration for Vmax. I should have 10x the Km for the highest substrate measurement to get a good curve, right?

Also, my Km is exactly 10 times lower than the literature value.

Furthermore, the specific activity in U/mg I measured is 225 times lower than the advertised minimum number Sigma Aldrich put on their product.I also tried to solve for a minimal difference between the sum the square of all the differences for the Michaelis-Menten formula to generate the Km and Vmax that best create a curve that fit my data points.

I should be at pseudo-Vmax when I take a substrate concentration 20x the Km. But for the given literature value, I am only at 3x the Vmax concentration at the highest data point.

The noise in the measurements doesn't seem too bad. It doesn't seem to be anywhere near big enough to be causing this issue.

I thought Km values were supposed to be the same for a certain enzyme under any circumstance where Michaelis-Menten is a good approx. and that Vmax only depends on enzyme concentration.

I checked my calculations again and again and I can check them again, but that doesn't see to move me forward.

I am supposed to be able to explain my results and explain the problem when I don't get literature values.

I am baffled. Is MM really so hard to apply, hard to measure? Am I missing something?

 

[epenguin]

It is hard and almost impossible to advise without seeing your data with properly labelled graphs plus a table of the raw data, any constants you are using, e.g. absorbances, and I frankly wouldn't mind you put up the stiructure of the various substances.(not ethanol and NAD though.) and explain what the reaction actually is.
For the comparisons of your data and ΣAldrich's and literature are they really determined in same conditions, what buffer, pH and temperature did you use and do you know how fresh the enzyme was, did you dilute or treat it anyhow? After which - your parameters are the ones you, not anyone else, measure, but don't expect that much reproducibility between labs.

 

[Almeisan]

I got an 8.5 on the report. Maybe they were diluted samples. I didn't really expect an answer in time or anyone to go through the data. Was just to confused when I posted this.

 

[epenguin]

Sorry I just happened not to have opened the biology section in these days. Might have suggested something but without seeing data and being able to dialogue I could not have. So all resolved and understood now?