Mechanism of Phosphorylation and Dephosphorylation
Covalent enzyme modification most commonly occurs via reversible phosphorylation. This process is tightly controlled by two functionally opposing groups of regulatory enzymes:
- Protein kinases — responsible for phosphorylation. They transfer a phosphate group from ATP to specific OH-groups of amino acid residues within target proteins.
- Phosphoprotein phosphatases — carry out dephosphorylation, removing the previously attached phosphate group.
The balance between the activity of these two enzyme groups ensures fine and reversible tuning of metabolic pathways within the cell. A deep understanding of this process requires studying the EC nomenclature classes of these enzymes and their strict substrate specificity.
Structure of Protein Kinase A (PKA)
A classic example of an enzyme regulated by and involved in covalent modification is Protein Kinase A (PKA). It is a cAMP-dependent enzyme (activated by cyclic 3',5'-AMP).
In its inactive state, PKA exists as a complex tetramer composed of four subunits:
- 2 regulatory subunits (R).
- 2 catalytic subunits (C).
This inactive complex is designated by the formula $R_2C_2$. Crucially, as long as the enzyme remains in this intact tetrameric form, it possesses absolutely no catalytic activity.
Mechanism of PKA Activation
The transition of Protein Kinase A from an inactive to an active state involves several sequential steps:
- Ligand Binding. The regulatory subunits (R) feature specific binding sites for cAMP molecules.
- Stoichiometry. Exactly 2 molecules of cAMP bind to each of the two R-subunits. Thus, a total of 4 cAMP molecules are required per tetramer.
- Conformational Changes. Upon cAMP binding, the spatial structure of the regulatory protomers changes.
- Dissociation. The $R_2C_2$ tetrameric complex loses stability and dissociates.
- Result. Two independent and fully active catalytic subunits (C) are released into the medium.
Active PKA can now perform its function: the phosphorylation of target proteins. The catalyzed reaction scheme is as follows: $E-OH + ATP \xrightarrow{PKA} E-O-PO_3H_2 + ADP$ (where E denotes the substrate protein).
Reversibility of Activation
The regulatory system could not be efficient without a mechanism to return to the baseline state. The PKA activation process is fully reversible.
When the intracellular signal concentration drops, cAMP molecules dissociate from the regulatory subunits. This immediately triggers the re-association of the free R- and C-subunits. They recombine to form the original inactive tetrameric complex, and target phosphorylation ceases.