The activity of a single-subunit enzyme that is controlled by negative feedback can at most drop from 90% to 10% when the concentration of an inhibitory ligand that it binds increases by a factor of 100. This is the maximum decline that can occur. Because the majority of enzymes that are activated or inactivated by ligand binding are composed of identical symmetrical subunit assemblies, this type of response does not appear to be precise enough for optimal cell control. This is due to the fact that the majority of enzymes that are activated or inactivated by ligand binding. With this design, the attachment of a ligand molecule to a specific site on a single subunit might potentially stimulate an allosteric shift in the entire assembly. This shift would make it easier for the neighboring subunits to bind the same ligand. Because of this, a cooperative allosteric transition takes place, which makes it possible for the entire assembly to be switched from a conformation in which it is almost entirely active to one in which it is almost entirely inactive (or vice versa) with only a moderate shift in the amount of ligand that is present in the cell.

When it comes to making a cooperative "all-or-none" transition, all proteins, regardless of whether or not they are enzymes, adhere to the same set of regulations. As a result, they are necessary for successful O2 uptake and release by hemoglobin in human blood, for instance. However, in the case of an enzyme that produces symmetric dimers, it is possible that they are the easiest to conceptualize. The binding of the first inhibitory ligand molecule to the dimer disrupts a connection that was energetically beneficial between the two identical monomers that make up the dimer, which makes the binding process extremely difficult. However, because the binding of this ligand restores the energetically favourable monomer-monomer interactions of a symmetric dimer, it is now more likely for a second inhibitory ligand to attach (this also completely inactivates the enzyme).

The regulation of proteins is impacted by a variety of factors in addition to the reversible binding of other molecules. Eukaryotic cells typically use a second method to regulate the activity of a protein, which is the covalent attachment of a smaller molecule to one or more of the amino acid side chains of a protein. This method can also be used by prokaryotic cells. The incorporation of a phosphate group is the regulatory modification that occurs in higher eukaryotes with the greatest frequency. For this reason, we will use protein phosphorylation to highlight some of the fundamental notions linked to the control of protein function by modifying the side chains of amino acids. Specifically, we will focus on the role that phosphorylation plays in this process.

Phosphorylation has the potential to have a major effect in three different ways on the protein that has been altered. To begin, the addition of a phosphate group to a protein that is catalyzed by an enzyme can significantly alter the structure of the protein. This can happen, for instance, when the positively charged amino acid side chains are drawn to the addition of the phosphate group. This is due to the presence of two negative charges within each phosphate group. This could then have an effect on how additional ligands attach to the surface of the protein, which would result in a major change to the activity of the protein. Once the phosphate group has been removed by a second enzyme, the protein reforms into its original structure and resumes its prior level of activity.

Second, there is a possibility that an attached phosphate group will contribute to the formation of a structure that can be recognized by the binding sites of other proteins. As was stated before, the SH2 domain affixes itself to a short peptide sequence that possesses a phosphorylated tyrosine side chain. Proteins can be attached to phosphorylated peptides in other protein molecules with the assistance of over ten additional common domains, each of which recognizes a phosphorylated amino acid side chain in a unique protein context. This allows proteins to interact with phosphorylated peptides in other protein molecules. Third, the presence of a phosphate group might mask a binding site that would ordinarily connect two proteins, which results in the disintegration of the connections that occur between the proteins in question. As a consequence of this, the phosphorylation and dephosphorylation of proteins are frequently responsible for dictating the rules that control the regulated assembly and disassembly of protein complexes.

Reversible protein phosphorylation in eukaryotic cells has a role in the regulation of enzyme activity, protein structure, and cellular localization, among other cellular processes. Because of the stringent control, it is assumed that more than one-third of the 10,000 or more proteins found in a typical mammalian cell are phosphorylated at any given time, typically with more than one phosphate. Phosphate groups are regularly added to and removed from specific proteins in response to signals that specify some change in the state of a cell. This was a phenomenon that might be anticipated. Many of the signals that regulate cell-cell interactions are transmitted from the plasma membrane to the nucleus via a cascade of processes involving protein phosphorylation. For example, the complicated sequence of events that take place during the division of a eukaryotic cell is timed in this manner for the most part.

Protein phosphorylation refers to the process in which the terminal phosphate group of an ATP molecule is transferred to the hydroxyl group of a serine, threonine, or tyrosine side chain on a protein molecule. Because of the significant amount of free energy that is generated during the process of breaking the phosphate-phosphate link in ATP to form ADP, which is catalyzed by a protein kinase, this reaction is practically unidirectional. This is due to the fact that the phosphate-phosphate link in ATP is broken to form ADP. Protein phosphatases are the enzymes that are responsible for the dephosphorylation reaction, which is the removal of phosphate from a protein. There are many different protein kinases that can be found in cells. Each of these protein kinases is responsible for phosphorylating a different protein or group of proteins. Some protein phosphatases are highly selective and can only remove phosphate groups from a single protein or a small number of proteins, whereas other protein phosphatases can function on a wide variety of proteins and are directed to specific substrates by regulatory subunits. Protein phosphatases are found in a variety of forms. The current state of phosphorylation and therefore the activity of a protein is determined by the relative activities of the protein kinases and phosphatases that are responsible for the change.

The protein kinases found in eukaryotic cells are members of a very large family of enzymes that share a catalytic (kinase) sequence that is no more than 290 amino acids long. These enzymes are responsible for the phosphorylation of proteins found in eukaryotic cells. The many members of the kinase family are distinguishable from one another at either end of the kinase sequence, and short sequences of amino acids are typically inserted into loops located inside it. Because of the presence of certain of these extra amino acid sequences, each kinase has the ability to identify the specific group of proteins that it phosphorylates, or it can attach to specific structures in order to localize itself in specific regions of the cell. It is possible for the activity of each kinase to be activated and deactivated in response to a number of diverse signals since the activity of the kinases is controlled by other protein components.

By comparing the number of amino acid sequence changes that have occurred between the various members of a protein family, it is possible for us to generate a "evolutionary tree" that is thought to depict the pattern of gene duplication and divergence that resulted in the formation of the family. This pattern is believed to have resulted in the formation of the family. Kinases that perform analogous functions are typically located on the same branch of the tree. For instance, the protein kinases that are involved in cell signaling and that phosphorylate tyrosine side chains are all clustered together in the upper left corner of the tree. The remaining kinases phosphorylate either a serine or a threonine side chain, and several of them are grouped into clusters that appear to correspond to their functions, such as the regulation of the cell cycle, the amplification of intracellular signals, and the transmission of transmembrane signals.

As a consequence of the collaborative efforts of protein kinases and protein phosphatases, the protein phosphate groups that are found on proteins undergo a continuous cycle of turnover. The faster the cycle, the quicker a population of protein molecules can change its state of phosphorylation in response to a sudden change in its phosphorylation rate. Although such phosphorylation cycles may appear to be wasteful, they are necessary for phosphorylated proteins to rapidly switch from one state to another. This phosphorylation cycle is powered by the liberated energy that is produced by the hydrolysis of ATP, of which one molecule is utilized for each phosphorylation event.