Recently I am stuggling to improve the kinetics of an artifical enzyme. I expressed this enzyme in E.coli and then test it's kinetics properties.
I noticed that if I pick single conies for testing, there will have a variation in both reaction rate and maxium reaction. indicating in fig.1 (the y axis represent the product that have been formed and the x axis represent the time in seconds.) 4 different conies have been picked and they all contain the same plasmid that transfection at the same time and same procedures. However there is huge differeces in the reaction rate and maxium reaction.
Then I wonder if it's due to the different conies would fold the protein differently, so I did another test by add multiple conies (actually all conies on one dish) into my culture medium. And then I test this mixed enzyme with different substrate concentration to test the affinity and kinetics at the same time. fig.2 (different color represent different concentration; the dash line represent a Imaginary limitation)
The problem that makes me wonder is that: what might be the reason for this reaction have a rate limitation?
I have few hypothesis about this phenomeon:
1. based on the Imaginary rate limitation; there might have steric effects preventing the binding of the substrate. (but I don't have see enough enzymatic reaction curve that have steric effects)
2. based on the varation between conies; this artifical enzyme might have many different ways of folding (I mean this enzyme would have many different prefered structures in different bacteria cells). maybe bactria from the same coniey would prefere similar stucture? and some stucture have better enzymatic performance, others do not.
I am really appreaciarte your reading and would be very happy to receive any response.
Hi there,
do you actually perform cell lysis and clarification by centrifugation prior assay?
It seems to me there is an inactivation process in your kinetics in figure 1 otherwise the end point should be the same for every clones.
Since you frequently refer to colonies ("conies"), it seems that you are not growing a culture of the cells and purifying the enzyme, just extracting the enzyme from the cells in the colony. In that case, there is not much point in studying the kinetics of the enzyme because it has not been purified. The signal obtained may be due to a mixture of multiple enzymes. The amount of the enzyme obtained from a colony may depend on factors such as how many cells are in the colony, the age of the colony, and how well the enzyme were extracted from each colony.
Generally speaking, an enzyme has a single overall conformation. It will not have different structures in different colonies. It might be expressed to different levels in different colonies. Some of it may be in an insoluble form due to failure to fold properly, and the proportion of insoluble, inactive protein may differ between colonies.
If you want to study this enzyme's kinetic properties, you really should purify it.
Dear Dr. Shapiro;
Thank you! I do agree with your point that enzymes needs to be purified before testing kinetics. I might do things too hury that forgot to check it's viability.
And I shall follow your suggestion to perform purification together with use higher centrifugation speed next time.
(I do have a spelling problem when I am too exciting about something. Thank you for pointing out I shall spell words correctly in the future)
really appraciate your answer!
I would assume, that you have simple reached a conditions of pseudo first order reaction conditions. Simply speaking, if you have reaction between two compounds and their concentrations are comparable, the reaction rate depends on both reagents concentrations. But if you start to increase concentration of one of the reagents (like in 100 times), the reaction rate becomes independent of its concentration.
Thanks for all, I figured out later that this problem is cause by the solution I choosed(PBS), that enzyme binding with phospate groups in PBS seems the possible factor which caused such slow kinetics. I switched to 50mMTris and 150mM NaCl then observed extremly fast kinetics.
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