Models of Allosteric Activity

Several theories have been developed to describe allosteric transitions. They may be generally grouped into the following three classes:

> 1. Sequential models: The prototype for the models that describe allosteric transitions is the sequential model of Koshland, Nemethy, and Filmer (KNF) (Figure 7.10a). The KNF model assumes that the subunits can change their tertiary conformation one at a time in response to binding of oxygen. Cooperativity arises because the presence of some subunits carrying oxygen favors the strong-binding state in adjacent subunits whose sites are not yet filled. Thus, as oxygenation progresses, almost all the sites become strong-binding. Such models are characterized by the existence of molecules with some subunits in the weak-binding state and some in the strong.

> 2. Concerted models: According to the theory of Monod, Wyman, and Changeux (MWC) (Figure 7.10b), the entire hemoglobin tetramer exists in an equilibrium between two forms - the deoxy (T) state, in which all subunits in each molecule are in the weak-binding conformation, and the oxy (R) state, in which all subunits are in the strong-binding form. (The symbols "T" and "R" stand for "tense" and "relaxed"; the significance of this is explained in the next section.) An equilibrium between the T and R states is presumed to exist, and partial oxygenation shifts that equilibrium toward the R state. The shift is a concerted one (different from the sequential model above), so that mixed molecules with some subunits in the weak-binding state and some in the strong-binding state are specifically excluded.

> 3. Multistate models: Neither the KNF nor the MWC model exactly explains the allosteric behavior of proteins, including hemoglobin. Consequently, more complex models have been devised. Most such models retain the MWC concept of a concerted switch in conformation but involve more than two states for the entire molecule.

Transitions and Subunit-Subunit Interaction - Transition from the deoxy to the oxy conformation involves major changes in the details of subunit-subunit interaction. Note the region in Figure 7.12b, to the lower left, where the 2 subunit interacts with the 1 chain. In the deoxy form, the C-terminus of 2 (residue 146) lies atop the C helix of 1 (residues 36–42) and is held in this position by a network of hydrogen bonds and salt bridges. His 97 in the FG corner of 2 is pushed against the CD corner of 1, between Thr 41 and Pro 44. In the oxy form, rotation and sliding of the subunits have pulled the C-termini of chains away from contacts (Figure 7.12b). The salt bridges and hydrogen bonds holding the C-terminus have been broken, and His 97 of 2 now lies between Thr 38 and Thr 41 of 1. Because of the symmetry of the structure, an exactly equivalent set of changes occurs at the 2 1 interface. The molecule has, as it were, "switched" and clicked into a new set of interactions. In the process, a number of strong interactions (those involving the C-termini in particular) have been broken. The looser conformation is called relaxed (R). The energy price for the change from the T state to the R state is paid by the binding of O 2 to the molecule. Once the O 2 has departed, the molecule will naturally fall back into its lower-energy deoxy conformation. This tighter conformation is called tense (T).

O 2 Binding and Molecular Switching - Figure 7.13 shows the relationship of His F8 and the neighboring Val (FG5) to the heme in deoxyhemoglobin. The iron atom in the deoxy conformation is a bit above the mean heme plane, but also the heme itself is not quite flat. Instead, it is distorted into a dome shape. Furthermore, in both deoxymyoglobin and deoxyhemoglobin, the axis of His F8 is not exactly perpendicular to the heme but is tilted by about 8°. When oxygen binds to the other side, it pulls the iron atom a short distance down into the heme and flattens the heme (Figure 7.13b,c). This change causes molecular rearrangement, for without it, both the -hydrogen of His F8 and the side chain of Val FG5 would be too close to the heme. Consequently, the histidine changes its orientation toward the perpendicular, shifting the F helix and the FG corner in the process. This movement in turn distorts and weakens the whole complex of H bonds and salt bridges that connect FG corners of one subunit with C helices of another. Consequently, the rearrangement shown in Figure 7.12 occurs.

In the simplest terms, the binding of O 2 pulls the iron a fraction of a nanometer into the heme, producing a lever effect which results in a much larger shift in the surrounding structure, particularly at the critical – interfaces. These changes constitute a rearrangement of the tertiary structure of each subunit upon oxygen binding.

A major rearrangement of the quaternary structure also occurs between the fully deoxy and fully oxy (T and R) states of the entire tetramer. How are the tertiary and quaternary structural changes connected? Figure 7.15 shows that the changes in tertiary structure that accompany oxygen binding can be tolerated up to a certain point before the T-R switch occurs. Specifically, whenever one site is occupied on each of the two - dimers, the molecule as a whole adopts the R quaternary structure.

Thus, hemoglobin obeys neither the KNF nor the MWC model completely but follows a novel path containing features of both models. This more recent model does not mean that the earlier models are generally incorrect. Allosteric proteins exist that appear to follow the MWC model almost exactly.

Other Allosteric Effectors - Cooperative binding and the transport of oxygen are only part of the allosteric behavior of hemoglobin. As oxygen is utilized in tissues, carbon dioxide is produced and must be transported back to the lungs or gills to be expelled from the organism. Accumulation of CO 2 lowers the pH in erythrocytes through the bicarbonate reaction,

CO 2 + H 2O <-> HCO 3- + H+

This reaction in erythrocytes is catalyzed by the enzyme carbonic anhydrase. High demand for oxygen, especially in muscle involved in vigorous activity, can result in oxygen deficit. A consequence of oxygen deficit is the production of lactic acid, which also lowers the pH. The falling pH in tissue and venous blood signals a demand for more oxygen.

Hemoglobin functions efficiently to meet these requirements. It does so through its allosteric transition between structurally different high-affinity and low-affinity states. Carbon dioxide, protons, and other substances all affect hemoglobin and promote allosteric changes.

See also: Hemoglobin Allostery, The Bohr Effect, Carbon Dioxide and Hemoglobin, Bisphosphoglycerate and Hemoglobin

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