Trying to master steady-state kinetics using a wild-type version of an enzyme my lab works with (PGM-1) against various concentrations of G1P (25uM-800uM). Obviously the protein concentration is being kept constant in each well. Ideally the activity of the enzyme should be leveling off/decreasing with the higher level of G1P (800uM) but the enzyme activity seems to be steadily increasing every time I attempt the assay.
The experiment is done using the help of a plate reader. To increase efficiency I'm using a multi channel pipette to initiate the reaction and place the plate into the reader as fast as possible. I've tried using fresher enzyme which has only helped slightly. I've also tried using a higher concentration of G6PDH. The only other thing that I haven't tried is using fresh DTT, but I don't want to make new DTT batches unless I absolutely have to. I'm having trouble thinking of what else to change, if anyone has any suggestions that would be much appreciated!
If the initial rate keeps increasing as you increase the substrate concentration, it means that the Km for that substrate is much higher than the concentrations that you are using, under the specific set of conditions you are using (pH, salts, temperature, etc.), assuming there are no artifacts...
Since you are using a coupled enzyme assay, check the rate when you leave out PGM-1 from the reaction. Sometimes the substrate of the primary enzyme can also act as a substrate for the coupling enzyme, or it can be contaminated with the substrate for the coupling enzyme. If you see a significant rate without PGM-1, you can subtract that rate from the rate with PGM-1 to get the rate due to PGM-1 alone.
If the initial rate keeps increasing as you increase the substrate concentration, it means that the Km for that substrate is much higher than the concentrations that you are using, under the specific set of conditions you are using (pH, salts, temperature, etc.), assuming there are no artifacts...
Since you are using a coupled enzyme assay, check the rate when you leave out PGM-1 from the reaction. Sometimes the substrate of the primary enzyme can also act as a substrate for the coupling enzyme, or it can be contaminated with the substrate for the coupling enzyme. If you see a significant rate without PGM-1, you can subtract that rate from the rate with PGM-1 to get the rate due to PGM-1 alone.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes