Mechanism of phosphorylation and dephosphorylation
Covalent modification of enzymes most commonly occurs via reversible phosphorylation. This process is strictly controlled by two groups of regulatory enzymes with opposing actions:
- Protein kinases — responsible for phosphorylation. They transfer a phosphoric acid residue from an ATP molecule to specific OH groups of amino acid residues within target proteins.
- Phosphoprotein phosphatases — carry out dephosphorylation, i.e., the removal of the previously attached phosphate group.
It is the balance between the activity of these two groups of enzymes that ensures the fine and reversible tuning of metabolic pathways in the cell. For a deep understanding of the process, it is necessary to study the classes of these enzymes according to the EC nomenclature and their strict substrate specificity separately.
Structure of Protein Kinase A (PKA)
A classic example of an enzyme regulated by and participating in covalent modification is protein kinase A (PKA). By type, it is a cAMP-dependent enzyme (dependent on cyclic 3',5'-AMP).
In its inactive state, PKA is a complex structure — a tetramer consisting of four subunits:
- 2 regulatory subunits (R).
- 2 catalytic subunits (C).
This inactive complex is conventionally designated by the formula $R_2C_2$. It is important to note that as long as the enzyme exists as such an intact tetramer, it possesses absolutely no catalytic activity.
Mechanism of PKA activation
The transition of protein kinase A from the inactive to the active state involves several consecutive steps:
- Ligand binding. Regulatory subunits (R) possess special sites for binding cAMP molecules.
- Stoichiometry. Exactly 2 cAMP molecules bind to each of the two R-subunits. Thus, a total of 4 cAMP molecules are required for the entire tetramer.
- Conformational changes. As soon as cAMP binds, 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.
Now the active PKA can perform its function — protein phosphorylation. The scheme of the catalyzed reaction is as follows: $E-OH + ATP \xrightarrow{PKA} E-O-PO_3H_2 + ADP$ (where E denotes the substrate protein).
Reversibility of the activation process
The regulatory system could not be effective without a mechanism to return to the initial state. The PKA activation process is completely reversible.
When the intracellular signal concentration drops, cAMP molecules dissociate from the regulatory subunits. This immediately triggers the reverse association of free R- and C-subunits. They reunite to form the original inactive tetrameric complex, and the target phosphorylation process ceases.
Briefly: answers to topical questions
Which specific amino acid residues are phosphorylated by protein kinases?
Protein kinases phosphorylate specific OH groups of amino acid residues in target proteins. These amino acids include:
- Serine — is phosphorylated by protein kinase A and undergoes transautophosphorylation in growth factor receptors.
- Threonine — is a target for phosphorylation by protein kinase A and MEK kinase.
- Tyrosine — undergoes autophosphorylation in growth factor and insulin receptors, and is also phosphorylated by MEK kinase.
To which EC nomenclature classes do protein kinases and phosphoprotein phosphatases belong?
According to the EC nomenclature, these enzymes belong to two different classes.
- Protein kinases — belong to the class Transferases (subclass phosphotransferases). They catalyze the transfer of a phosphoric acid residue from an ATP molecule to a substrate protein.
- Phosphoprotein phosphatases — belong to the class Hydrolases (subclass phosphatases). They carry out a hydrolysis reaction, removing the phosphate group from the enzyme to form inorganic phosphate.
To memorize the composition of inactive PKA, use the R₂C₂ tetramer formula: two Regulatory and two Catalytic subunits. They are active only when they dissociate under the action of 4 cAMP molecules.
Class and exam questions
What is the core essence of covalent modification of enzymes?
The core essence lies in the rapid and reversible addition or removal of a chemical (most often phosphate) group to the enzyme molecule, which alters its activity.
How many cAMP molecules are required to activate one molecule of protein kinase A?
Full enzyme activation requires 4 cAMP molecules — 2 molecules for each of the two regulatory subunits.
Does the $R_2C_2$ tetramer of protein kinase A possess catalytic activity?
No, in the form of the assembled tetrameric complex, the enzyme is inactive. Catalytic activity appears only after dissociation of the complex and release of the C-subunits.
What else to study on this topic
- Classification of kinases and phosphatases according to the Enzyme Commission (EC) nomenclature
- Substrate specificity of protein kinases and phosphoprotein phosphatases
- Detailed equations of phosphorylation and dephosphorylation reactions
- Medical aspects of enzymology and pathology of enzyme regulation
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