A single-subunit enzyme that is regulated by negative feedback can at most decrease from 90% to about 10% activity in response to a 100-fold increase in the concentration of an inhibitory ligand that it binds (Figure 3–59, red line). Responses of this type are apparently not sharp enough for optimal cell regulation, and most enzymes that are turned on or off by ligand binding consist of symmetric assemblies of identical subunits. With this arrangement, the binding of a molecule of ligand to a single site on one subunit can promote an allosteric change in the entire assembly that helps the neighboring subunits bind the same ligand. As a result, a cooperative allosteric transition occurs (Figure 3–59, blue line), allowing
Figure 3–57 Positive regulation caused by conformational coupling between two separate binding sites. In this example, both glucose and molecule X bind best to the closed conformation of a protein with two domains. Because both glucose and molecule X drive the protein toward its closed conformation, each ligand helps the other to bind. Glucose and molecule X are therefore said to bind cooperatively to the protein.
Figure 3–58 negative regulation caused by conformational coupling between two separate binding sites. The scheme here resembles that in the previous figure, but here molecule X prefers the open conformation, while glucose prefers the closed conformation. Because glucose and molecule X drive the protein toward opposite conformations (closed and open, respectively), the presence of either ligand interferes with the binding of the other.
a relatively small change in ligand concentration in the cell to switch the whole assembly from an almost fully active to an almost fully inactive conformation (or vice versa).
The principles involved in a cooperative “all-or-none” transition are the same for all proteins, whether or not they are enzymes. Thus, for example, they are critical for the efficient uptake and release of O2 by hemoglobin in our blood. But they are perhaps easiest to visualize for an enzyme that forms a symmetric dimer. In the example shown in Figure 3–60, the first molecule of an inhibitory ligand binds with great difficulty since its binding disrupts an energetically favorable interaction between the two identical monomers in the dimer. A second molecule of inhibitory ligand now binds more easily, however, because its binding restores the energetically favorable monomer–monomer contacts of a symmetric dimer (this also completely inactivates the enzyme).
As an alternative to this induced fit model for a cooperative allosteric transition, we can view such a symmetric enzyme as having only two possible conformations, corresponding to the “enzyme on” and “enzyme off” structures in Figure 3–60. In this view, ligand binding perturbs an all-or-none equilibrium between these two states, thereby changing the proportion of active molecules. Both models represent true and useful concepts.
Proteins are regulated by more than the reversible binding of other molecules. A second method that eukaryotic cells use extensively to regulate a protein’s function is the covalent addition of a smaller molecule to one or more of its amino acid side chains. The most common such regulatory modification in higher eukaryotes is the addition of a phosphate group. We shall therefore use protein phosphorylation to illustrate some of the general principles involved in the control of protein function through the modification of amino acid side chains.
A phosphorylation event can affect the protein that is modified in three important ways. First, because each phosphate group carries two negative charges, the enzyme-catalyzed addition of a phosphate group to a protein can cause a major conformational change in the protein by, for example, attracting a cluster of positively charged amino acid side chains. This can, in turn, affect the binding of ligands elsewhere on the protein surface, dramatically changing the