During standardization of an enzyme to study its activity, it showed a linear trend of increase up to 4 h then it gets stabilized. For the first time I got such an enzyme to have its maximum activity after 4 h incubation with its substrate in unlimited quantity. How the curve is justified?
The enzyme is activated, either by the product (if the product accumulates under the conditions of the assay), or by self-processing (if., e.g., the enzyme is a protease, or a kinase...) or simply by incubation under conditions (temperature, pH, ionic strength etc) different from the storage conditions. The first two options seem more convincing to me.
To sort out the different possibilities, assuming it is worth doing, you'll need to do a bunch of controls,.For example, to assess the possibility of product activation, you can check whether the time needed to reach maximum activity depends on the overall enzyme concentration, or on the addition of product at the very beginning of the reaction. Also, you may pre-incubate the enzyme for a few hours under the assay conditions, but omitting the substrate, and see if preincubation is enough to bring about activation...
Thanks Dr. Peracchi for your answer. However, the curve is linear for its activity in the first 4 hours. So such result was forcing to think that the enzyme needs time to be fully activated by conditions or substrates. Any other idea?
Hi Biswaranjan, you just reiterate that achieving full activation takes four hours. .This, however, was already clear from your question. Products buildup is necessarily time-dependent, self-processing may be slowr and certain conformational changes can require a long timescale. Thus the time required for activation, by itself, does not indicate what are the basis of the phenomenon. I think the controls I suggested would help understand what is going on, but I could give better suggestions if I knew more about your enzyme and assay system, and maybe see some plot of activity vs. time,
Hi Biswaranjan,
It isn't clear to me whether you are describing an enzyme reaction rate that is getting faster with time (activation, as discussed by Alessio, indicated by an upward curving progress curve), or a constant rate of product accumulation (a linear progress curve, at least at the beginning).
If it is the latter, then your question is why does it slow down even though there is, as you say, an unlimited amount of substrate? There are at least 2 possible reasons: (1) the enzyme is unstable and loses activity after a while, or (2) the forward reaction is inhibited by a product of the reaction.
Some enzyme reactions are sufficiently reversible that their reactions reach an equilibrium before the substrate is consumed. In some cases, the product is so similar to the substrate that it acts as a a potent inhibitor.
It would help to know more about the system you are using. For example, how is it that the substrate is "unlimited"? Is the reaction reversible? Is there just one substrate, or more than one?
Dear Dr. Peracchi and Dr. Shapiro, The enzyme system is HRP mediated H2O2 determination system using HVA and H2O2 as substrate (in unlimited quantity). During standardization I got such results. I must define that I could not minimize the time of incubation of the above reaction rate with high amount of substrates. For your kind perusal I am attaching the file. Thanks
Thank you for showing the data graph, Biswaranjan. Now it is clear to me that the situation you are seeing is an increasing rate of product formation with time. How are you detecting the product of the reaction?
Biswaranjan the kind of enzyme you are handling is involved in a highly spontaneous reaction. In such kind of enzyme assays a kinetic mode of analysis is followed with an intervals of 30 seconds and the monitoring is done for 3 min or so. From your graph is appears that you have added the substrate in such a high quantity that the rate of reaction is linear for 4 hours. Thereafter the substrate becomes limiting or there is certain kind of feedback inhibition and you get a flat curve which is very common in enzyme kinetics. I would suggest you to perform a kinetic assay and see the rate of change in Absorbance per unit time. I am very much sure you will get the right enzyme activity... Good luck...
Hey there all, thank you for your interaction and sorry for the delay in updating. Actually I measured the product in a fluorimeter. After conducting certain experiments, now the answer is little clear to me that the enzyme is fully active up to 4 hours and after that it starts degrading in a linear manner. As a result the product formation rate is not decreased rather remain in a constant region. So the optimum time for the activity can be assayed within any time up to 4 h of incubation. Am I doing correctly.
Biswaranjan, you are saying that the reaction rate is constant during the first 4 hours when you use a fluorescence measurement? The graph you showed earlier showed a rate that was increasing with time until it leveled off. Can you explain?
Hi Adam, The rate of product formation with the enzyme is constant so that the accumulation of the product increases linearly up to 4 hours. Then may be due the enzyme becomes slowly unstable, the accumulation of the product does not takes place in a linear manner rather it deceases. However, the net product formation rate remains the same for a fewer hours and then it decreases. That means if the enzyme is producing 4 products (suppose) in an hour, then the no of products would increase in the reaction mixture as 4, 8, 12 and 16 in the first 4 hours due to the constant activity of the enzyme. However, since the enzyme get deactivated slowly in the next few hours, instead of the product no. 20 in the 5th hour, it remained as 16. May be this happens for few hours and then the number deceases slowly to 14, 12, 10 etc. as the linear rate of product formation does not take place.
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