Question 5: Allosteric behavior of anolein * Analysis of sedimentation and SDS-P

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Question 5: Allosteric behavior of anolein * Analysis of sedimentation and SDS-PAGE data suggests that anolein is a heptamer (7 subunits) consisting of three copies of a protein with MW = 50 kD and four copies of a second protein with MW = 100 kD (ie . The intact heptamer, rather surprisingly, exhibits allosteric kinetics (i.e. a sigmoid shaped activity plot of Vo vs. [S]). Addition of glucose to the intact heptamer results in a shift of the sigmoid plot to the left, while addition of 2,3-BPG results in a shift to the right . Treatment of anolein with 500 mM NaCl, followed by sucrose gradient centrifugation, results in two peaks with apparent MW- 150 kD and MW - 400 kD. The MW 150 kD peak has no discernible enzymatic activity, while the 400 kD peak exhibits full enzymatic activity. This activity, however, is no longer affected by glucose or 2,3-BPG What is the function of the 150 kD fraction of anolein? Why does it not have enzymatic activity? Why does the 400 kD fraction not exhibit allosteric kinetics, but the intact heptamer does? What type of kinetics would you expect for the isolated 400 kD fraction? Sketch the expected activity plot for this fraction Describe the allosteric behavior of the intact heptamer, assuming the concerted model. Include in your explanation the following features: effect of glucose, effect of 2,3-BPG, T state, R state, T-R equilibrium sketch the model a) b) c)

Explanation / Answer

a. 150 kDa fraction does not have an active site due to which it does not exhibit enzymatic activity. It is rather allosteric in nature and might bind to molecules altering the overall conformation and causing allostery of the enzyme. It may also serve as a structural scaffold for overall control of enzymatic activity(allosteric in nature).

b. The 400 kDa fraction exhibits enzymatic activity due to the presence of catalytic site. However, it does have an allosteric domain/ region in its 400 kDa structure but when it interacts with the other subunit , allosteric activity on a structural and functional perspective gets active.

The curve will not be a sigmoidal and rate of a reaction keeps increasing till all the active sites are filled with the substrate. Once that happens a saturation for rate of reaction occurs and remains the same , even though the substrate concentration is increasd.

C. Anolein when binds to its substrate which is glucose it is in R state due to conformational changes, thus enzymatically active. When it is without a substrate, it is in T state , a state where no enzymatic activity occurs. Again upon binding to an allosteric regulator say 2,3 bpg to R state conformation of the protein changes and it attains a T state. There is a transition state between R and T that cannot be captured using enzymatic assays but can be captured through deuterium exchange mass spectrometry.

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