Based on the information and the graph given, what sort of conclusions can be ma

Question

  

Based on the information and the graph given, what sort of conclusions can be made? What concepts can be applied to get more information for this problem?

I am attempting to create the Glycolytic pathway (Glcpyruvate) in vitro. I purify all the enzymes of the pathway, the co-factors required, NAD+, ADP, ATP and P. I then mix all these components together and add Glucose. I then measure the ratio of Pyruvate:Glucose in the mixture over time. My data is shown in the graph. At the point marked "X" in the graph, I added a non-protein substance to the pathway. Time

Explanation / Answer

Therefore based on the above knowledge and the trend of the curve given, it is clear that the glycolysis reactions first come to a standstill due to a limiting substrate or due to feedback inhibition. For example, a) glucose substrate may be limiting, b) increase in ATP levels can inhibit phosphofructokinase or pyruvate kinase. It is clear that addition of substance X feedforwards the pathway, e.g.: a) if glucose substrate was limiting, no more pyruvate will be produced, then the pyruvate : glucose ratio will remain unchanged till more glucose is added, resulting in an increase in the product: reactant ratio, until the substrate is once again limiting. b) Any non protein scaffold which can selectively sequester ATP, so that the high levels of this nucleotide does not inhibit the enzymes. (e.g.: bis(Zn2+-cyclen)-triethylbenzene).

If glucose is not limiting and even if ATP does not interfere with the reactions, then we can choose X to understand which enzyme has been selectively inhibited. If F-1,6-BP and not F-2,6-BP feedsforward the pathway, then the emzyme inhibited was pyruvate kinase. If F-2,6-BP or AMP restimulates the pathway, then the enzyme inhibited was phosphofructokinase. If X is glucose-6-phosphate, then it can be inferred that the hexokinase is not functioning properly or the enzyme turnover may be very low.

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