I want to find out the activity of certain enzymes in bacteria. I have seen papers in literature in which people have taken crude extract of cells and have done enzyme activity. Now, suppose enzyme 'A' converts substrate X to Y and at the same time enzyme 'B' converts Y to Z. In crude extract, both enzymes 'A' and 'B' will be present. If i want to find out activity of A, then i will have to add analogue of X but as soon as product Y will be formed, enzyme 'B' will convert it to next product. Then how will I be able to find out activity of A correctly? The product Y is getting converted to Z as soon as it is getting formed so how should I do enzyme activity experiments? Please clarify my doubts.
What you say is true, but most enzymatic reactions require more than one component. For example, ATP, NADH, or some other co-substrate. If the reaction catalyzes a hydrolysis, you can look for the product, which should accumulate unless the co-substrate for the next step is present.
You can use general inhibitors for different classes of enzymes. You can use alternative substrates that may only undergo one reaction or that may not get processed as well by down stream enzymes. You can follow product formation. There should be an initial rate followed by a plateau. If another enzyme is using the product, the rate may not increase linearrily with time. It might start to go up, but then go back down.
Most importantly, One can add the product for the first enzyme, assuming you know what the reaction is for enzyme A, and see if it gets consumed. Then you will know if there is an enzyme B present.
This is what happens in coupled assays. If there is an excess of enzyme B, so that everything is converted without delay to Z, then the rate you see is dependent on X going to Y by enzyme A and all you need to do is measure the production of Z. If enzyme B is rate limiting, then when X goes to Y, Y will accumulate and you need to measure Y directly to get the rate of conversion.
Thanku for your suggestions..i think if i will analogue of the substrate for enzyme A then its product will not be acted upon by enzyme B because that will not be a natural product...
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