pCAP-Based Peptide Substrates: The New Tool in the Box of Tyrosine Phosphatase Assays (nih.gov)
My group is making a substrate-containing probe that has a lower Km than the substrate alone for a protein. The substrate has a very high Km to the protein, which is said to be not good. I don't understand this. Why do we need a substrate/probe with a lower Km for an enzymatic assay?
Will we get more accurate results? Will we need less substrate to achieve the same turnover?
See: https://en.wikipedia.org/wiki/Michaelis–Menten_kinetics
The Km represents the substrate concentration at which the enzyme works at half-maximal speed - it basically represent the "affinity" of the substrate to the enzyme. A small Km means that the reaction rate will approach the maximal rate at lower substrate concentration than if the Km is high.
As Annemarie Honegger said, lower Km means that the enzyme has higher affinity for the substrate and vice versa. Thus, you will need less of your probe for an assay than you would need of the original substrate. This may lead to a more cost-effective, and/or a more robust experimental setup. There is also likely a question on assay sensitivity and I imagine that your probe was designed in such a way that it allows better monitoring of reaction progress (compared to the original substrate).
Hi there,
Just answering your 3 questions very briefly:
Working with a modified substrate exhibiting a lower Km than the natural means you will have to use less to observe the same level of activity.
The results will be the same quality.
Yes for a same turnover number, you will have to use less of the substrate exhibiting the lower Km as Km is one of the determining parameter of Michaelis Menten equation.
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