I am measuring the production of NADH in a cell extract provided with glucose. If I measure the formation of a NADH over time, which is produced by the combined action of a chain of different reactions, catalyzed by different enzymes (HK to GAPDH in glycolysis), and I then plot V0 values according to substrate concentration, can I determine a Km and a Vmax value? And what will these values correspond to essentially? Km is normally an indicator of enzyme affinity for substrate, is the Km for a chain of reactions therefore dictated by the enzyme with the lowest affinity for its substrate?
Dear Max,
Of course you'll be able to measure a so-called Vmax and Km for the overall reaction. What it means exactly will be less clear. You'll measure a value for both constants which is composed of contributions of all the enzymes involved in the overall pathway with the most important contribution coming from the enzyme(s) that function(s) as the limited factor in the overall reaction. In the case of glycolysis the pathway has been mathematically modeled in great detail for yeast and trypanosomes. In the case of trypanosomes I can recommend to read the papers by @Barbara Bakker on the subject. You'll find them on ResearchGate in one of my projects (https://www.researchgate.net/project/Metabolic-control-analysis-and-regulation-of-glycolysis)
Good luck, Fred Opperdoes
I agree with Fred, if you perform the experiment you suggest, the Vmax will reflect the rate determining step/enzyme (which could possibly change over the concentration range you examine). Therefore, you will likely find the Vmax of this one rate determining enzyme. The km for NADH determined is likely to be a complex issue, and will likely not directly relate to that of GAPDH unless it is the rate determining enzyme/step. It would be much better to try and study the activity of this enzyme in isolation rather than in a pathway.
All the best, Phil
instead of trying Michaelis-Menten it is better to try Lineviewer bruck
Max, the Michaelis-Menten equation and the Lineweaver-Burk plot are two different presentations of the same correlation between the rate of an enzymatic reaction and the affinity of the enzyme for its substrate. The Michaelis-Menten equation represents a hyperbolic curve while the reciprocal Lineweaver-Burk plot represents a straight line. The latter is used for the rapid and precise estimation of Km and Vmax . For more info on these two plots I suggest to watch Dr Mungli's Youtube video on the subject
https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwj7qPiZz6LtAhWS3KQKHZA6AqsQwqsBMAN6BAgEEAs&url=https%3A%2F%2Fwww.youtube.com%2Fwatch%3Fv%3DECRn8Ox6vWY&usg=AOvVaw2A4FIw0vqg67vy8-5ENnFx
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