Chemical aspects of phosphoric acid
Now let's take a closer look at phosphorus from a chemical perspective. Phosphorus exists in the body as phosphoric acid (H₃PO₄), also known as orthophosphoric acid. Influenced by various environments such as pH, phosphoric acid exists in three forms in aqueous solution by losing hydrogen protons one at a time. When phosphoric acid loses all of its protons, it becomes a phosphate.
When two or three phosphoric acids combine, they form pyrophosphoric acid and triphosphoric acid, respectively, as depicted in the image below. Additionally, when two or three phosphates, which are the forms with hydrogen protons removed, combine, they form diphosphate and triphosphate, respectively.
Ester bond
At this point, it is necessary to briefly examine the esterification reaction in chemistry. An ester is a compound formed when an acid reacts with an alcohol. In chemistry, the term "alcohol" refers to organic compounds (R-OH) where the hydrogen atoms of a carbon chain are replaced by hydroxyl groups (OH). Simply put, you can think of it as a carbon atom with an OH group attached.
Taking carboxylic acid (R-COOH), which is the most representative acid, as an example, when carboxylic acid reacts with alcohol, an ester compound (R-COO-R') and water (H2O) are produced. If we denote the remaining hydrocarbon parts of the organic compounds as R and R' (represented as R1 and R2 in the diagram below), the formation of the ester compound can be briefly illustrated as shown in the diagram. The hydrogen from the carboxyl group combines with the hydroxyl group (OH) from the alcohol compound to form water, leading to the bonding of the two molecules. For reference, this process, in which two molecules combine to produce a new compound while generating one molecule of water is called a dehydration condensation reaction.
Let’s substitute phosphoric acid for the carboxylic acid in this ester bond. Instead of carbon, we place phosphorus, resulting in the structure R1-POO-R2, which we call a phosphoester bond. As shown in the diagram below, one phosphoric acid molecule shares its two hydroxyl (OH) groups with the hydroxyl groups of two sugar molecules, forming a phosphodiester bond. The 'di-' in the middle means two, indicating that there are two ester bonds. This bond forms the outer helical backbone of DNA and RNA. The diagram below illustrates how the phosphoric acid connects to the 3rd and 5th positions of the ribose sugars, resulting in the formation of the ester bond while releasing water.
Phosphodiesterase(PDE)
Of course, there are enzymes that break these bonds in reverse. Just as the bond is formed between phosphorus and hydroxyl groups with the release of water, phosphodiester bonds are broken by adding water to facilitate the reaction. This enzyme is called phosphodiesterase (PDE). It plays a central role in the signaling system through G-protein-coupled receptors (GPCR), which are the most representative cellular signaling pathways, by receiving external signals and regulating the appropriate levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) as secondary signalling messengers within the cell. This enzyme prevents excessive accumulation by breaking down cAMP and cGMP when they are present in excess within the cell. For more details, please refer to the section on GPCR related to intracellular signaling pathways. The ester bonds of phosphoric acid will be revisited later in the phosphorylation process of specific amino acids.
What is phosphorylation?
Now that we have examined the chemical aspects of proteins and phosphates, let's explore phosphorylation. Phosphorylation is the most common modification that occurs after protein synthesis is complete. Simply put, phosphorylation refers to the process of attaching a phosphate group to a substance. What happens when a phosphate group is attached to a protein? We know that the three-dimensional structure of a protein dictates its function. Since a phosphate group carries two negative charges, when it binds to a protein, it will repel areas of the protein that also carry a negative charge while attracting areas with a positive charge. Ultimately, these electrostatic attractions and repulsions will distort the shape of the protein, and this structural change will lead to altered protein function. If the protein is an enzyme, its activity may increase or decrease; if it is a transport protein, its transport capacity may change. Additionally, the stability and properties of the protein can also be affected. If a protein becomes phosphorylated and can no longer enter the nucleus, gene expression will be inhibited. Phosphorylation can also alter interactions with other molecules, including other proteins, lipids, DNA, and RNA. If multiple phosphate groups, which carry negative charges, are continually added, these changes will become even more diverse and pronounced.
Cells can phosphorylate target proteins to activate molecular activities, essentially turning the "switch" on, or deactivate them to turn the "switch" off. They can also enhance or inhibit active states, thereby regulating both the speed and quantity of activity. In short, phosphorylation serves as a control mechanism through which cells can manage and regulate various processes. Gene expression, cell proliferation, differentiation, apoptosis, signal transduction, immune regulation, and metabolic regulation are all processes that cells conduct on a daily basis. As we will explore in more detail regarding intracellular signaling, phosphorylation often follows a cascading process, where one phosphorylated protein phosphorylates the next protein, which in turn phosphorylates a downstream protein, resembling a waterfall flowing downward. Additionally, phosphorylation allows a single small signal from the outside to be amplified into a significantly larger amount of signals within the cell.
If any stage of this highly controlled mechanism encounters a problem, the consequences can be fatal. We can infer that defects in the phosphorylation process can lead to abnormalities in signaling pathways, resulting in the development of various serious diseases, including cancer, inflammatory diseases, neurological disorders, and neurodegenerative diseases. Research on various cancers has found that certain enzymes for protein phosphorylation are abnormally expressed. Therefore, in numerous studies aimed at treating cancer, the phosphorylation of proteins becomes an important focus of research.
Carbohydrates, lipids, and proteins can all be targets of phosphorylation; however, this discussion will focus specifically on protein phosphorylation. In this context, the substrate for phosphorylation is proteins. More precisely, phosphate groups are covalently attached to the side chains of amino acids. In the previous section, we briefly examined the basic structure of proteins. Phosphorylation primarily occurs on the side chains of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, which are attached to the common backbone shared by all amino acids. Although phosphorylation can also chemically occur on residues such as arginine (Arg), lysine (Lys), histidine (His), cysteine (Cys), aspartic acid (Asp), and glutamic acid (Glu),[1] the reason serine, threonine, and tyrosine are predominantly discussed in relation to phosphorylation is that they account for approximately 86.4%, 11.8%, and 1.8% of the occurrences, respectively, making up almost the entire proportion.[2]
These three amino acids have a hydroxyl group (OH) in their residues. Let's recall the ester reaction discussed above. Phosphoric acid is added to the hydrogen position of these hydroxyl groups and bonded. The figure below shows that when a phosphate group is bonded to serine, the hydroxyl group of serine is replaced by phosphate and becomes phosphorylated serine, i.e. phosphoserine.
These three amino acids all have a hydroxyl group (OH) in their side chains. Let's recall the ester reaction we discussed earlier. The phosphoric acid binds to the hydrogen position of these hydroxyl groups. The diagram below illustrates how, when a phosphoric acid attaches to serine, the hydroxyl group of serine is replaced by the phosphoric acid, resulting in phosphorylated serine, or phosphoserine.
Now, in the next article, let's look at kinases, enzymes that catalyze phosphorylation.
[References]
[1] Why nature chose phosphate to modify proteins
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3415839/
[2] Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10152985/
