Orientation of the binding site

Outline of the experiment to determine the orientation of a single binding site on unphosphorylated Il  

The first column indicates the possible orientations. Dynamic indicates that the site is not fixed at one side of the membrane. Whether or not binding would be measured is indicates by the + and — signs, respectively. The last line in the table gives the results from the actual experiment [30]. RSO, right-side-out ISO, inside-out. 

Unphosphorylated functioning according to Fig. 5 catalyzes facilitated diffusion of mannitol across the membrane. The same process has been reported for purified II reconstituted in proteoliposomes [70]. The relevance of this activity in terms of transport of mannitol into the bacterial cell is probably low, but it may have important implications for the mechanism by which E-IIs catalyze vectorial phosphorylation. It would indicate that the transmembrane C domain of Il is a mannitol translocating unit which is somehow coupled to the kinase activity of the cytoplasmic domains. We propose that the inwardly oriented binding site which is in contact with the internal water phase (Ecyt Mtl, see Fig. 5) is the site from where mannitol is phosphorylated when transport is coupled to phosphorylation. Meehan- 

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In conclusion, the steady-state kinetics of mannitol phosphorylation catalyzed by II can be explained within the model shown in Fig. 8 which was based upon different types of experiments. Does this mean that the mechanisms of the R. sphaeroides II " and the E. coli II are different Probably not. First of all, kinetically the two models are only different in that the 11 " model is an extreme case of the II model. The reorientation of the binding site upon phosphorylation of the enzyme is infinitely fast and complete in the former model, whereas competition between the rate of reorientation of the site and the rate of substrate binding to the site gives rise to the two pathways in the latter model. The experimental set-up may not have been adequate to detect the second pathway in case of II " . The important differences between the two models are at the level of the molecular mechanisms. In the II " model, the orientation of the binding site is directly linked to the state of phosphorylation of the enzyme, whereas in the II" model, the state of phosphorylation of the enzyme modulates the activation energy of the isomerization of the binding site between the two sides of the membrane. Steady-state kinetics by itself can never exclusively discriminate between these different models at the molecular level since a condition may be proposed where these different models show similar kinetics. The II model is based upon many different types of data discussed in this chapter and the steady-state kinetics is shown to be merely consistent with the model. Therefore, the II model is more likely to be representative for the mechanisms of E-IIs. 

As discussed above in Chapter 3, ellipsometry and quartz crystal microbalance (QCM) approaches provide a useful insight into the adsorption of both the supporting interfacial assembly and the proteins themselves. Beyond monitoring the adsorption dynamics and the structural integrity of the biomolecule, the orientation of the active site is of prime importance. For example, if the active site itself binds to the self-assembled monolayer, transport of the substrate or co-enzyme may be blocked. 

Figure 4. Two versions of the E,-E2 model illustrating the uncertainty with respect to the orientation of cation-binding sites in E,P and E2. The orientation is indicated by a dot (pointing upwards for cytoplasmic orientation and downwards for extracytoplas-mic orientation). In A the orientation of the cation-binding site with respect to the membrane changes simultaneously with a change in catalytic specificity of the ATP-binding/phosphorylation site. In B it is the phosphorylation that determines orientation of the cation binding site.

Quantum yield and fluorescence emission maximum are sensitive to the surrounding environment. This can be explained as follows. Fluorophore molecules and amino acids of the binding sites (in the case of an extrinsic fluorophore such as TNS, fluorescein, etc.) or the amino acids of their microenvironment (case of Trp residues) are associated by their dipoles. Upon excitation, only the fluorophore absorbs the energy. Thus, the dipole of the excited fluorophore has an orientation different from that of the fluorophore in the ground state. Therefore, the fluorophore dipole-solvent dipole interaction in the ground state is different from that in the excited state (Figure 7.18). 

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