I am working on a tetrameric enzyme, for which the velocity of reaction increases to a maximum and then starts decreasing with higher substrate concentrations without any apparent flat region. I would be grateful if someone could explain in a little detail.
It is important to note that in enzyme catalyzed reactions high substrate concentration as well as accumulation of product beyond a certain limit inhibit enzyme activity. The high substrate concentration likely to result the formation of such species of ES complex which is not active to be converted into E and P. On the otherhand, in case of regulatory enzymes the accumulation of product results into feedback inhibition of the first enzyme in a metabolic pathway. In your case, the enzyme is a tetrameric one and likely to be inhibited by second mechanism. If you separate enzyme from substrate/product (e.g. by dialysis or by other methods) then enzyme should get reactivated (if it is not quite unstable). You may also check that your substrate is not contaminted by some inhibitor, because with the increase of substrate concentration the concentration of inhibitor is also increasing and reaching a threshold to show suddenly a decline in reaction rate.
It is important to note that in enzyme catalyzed reactions high substrate concentration as well as accumulation of product beyond a certain limit inhibit enzyme activity. The high substrate concentration likely to result the formation of such species of ES complex which is not active to be converted into E and P. On the otherhand, in case of regulatory enzymes the accumulation of product results into feedback inhibition of the first enzyme in a metabolic pathway. In your case, the enzyme is a tetrameric one and likely to be inhibited by second mechanism. If you separate enzyme from substrate/product (e.g. by dialysis or by other methods) then enzyme should get reactivated (if it is not quite unstable). You may also check that your substrate is not contaminted by some inhibitor, because with the increase of substrate concentration the concentration of inhibitor is also increasing and reaching a threshold to show suddenly a decline in reaction rate.
For some multimeric enzymes, apparent substrate inhibition can arise from an equilibrium of functionally distinct multimers such as occur in the case of morpheeins.
Thanks to both of you for explaining.. and @Dr Jaffe: Just scanned through your paper on morpheeins.. Truly interesting and informative, thanks for introducing this concept to me :) My enzyme shows cooperativity and substrate inhibition. Is it possible for it to show morpheein-like character? Is there a basic technique that I can use to study this? Also, if you dont mind me asking, what is the physiological significance of such enzymes that show substrate inhibition or are morpheeins?
We have published extensively on the characteristics of proteins that use the morpheein model of allostery. The most simplistic description, and most of the references, can be found on the Wikipedia page we constructed. However, the bottom line is that this allosteric mechanism is implied by many different behaviors, but difficult to PROVE. For most of the behaviors, there are alternative explanations that do not require a dissociative equilibrium of architecturally distinct multimers. As for the physiological significance, I believe that the existence of alternate assemblies simply provides Nature with another level at which protein function can be regulated. This is also a way that one protein can have many functions (e.g. the moonlighting phenomenon). A wonderful example, though not an enzyme, is the Ebola virus VP40 protein as recently described by Erica Saphire and coworkers. In that case homo-dimers, linear homo-hexamers, and circular homo-octamers are each responsible for a different function in the virus life cycle.
Inhibition by excess of substrate. This can be caused by several mechanisms. For instance, the formation of dead-end SES complex in bisustrate reactions (two molecules of the same substrate are bound to the enzyme). It is caused when the concentration of the substrate with higher affinity by the enzyme is increased. The inhibition of peroxidases by excess of hydrogen peroxide is a good example..
You must have checked but an altered pH of the reaction mixture with excess substrate (or its contaminant) might account for the rate decline.
Read the following article, it would be interesting for you:
https://www.ncbi.nlm.nih.gov/pubmed/20414900
and a little bit older about product inhibition
DOI: 10.1016/0022-5193(83)90325-9
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