Phosphorylation

Phosphorylation is a chemical process in which a phosphoryl group (PO₃^2-) is added to an organic compound. In other words, phosphorylation meaning in chemistry is depicted as an organic process that involves the addition of a phosphorous group with an organic compound. For example, when phosphate is added to glucose, it becomes glucose monophosphate. In a similar manner, when phosphate is added to adenosine diphosphate it results in adenosine triphosphate. 

Phosphorylation plays a very important role in regulating protein function and transmitting various signals throughout the human body cell. Though it is predominantly observed in bacterial protein, it is considered more prevalent in eukaryotic cells. It has been observed at some point in time, one-third of the protein present in the human proteome are substrates of phosphorylation. Therefore, phosphoproteomics has evolved as a part of proteomics that focuses only on identifying and characterizing phosphorylated proteins. 

Other than this, phosphorylation helps in conserving much of the energy in food by the process of oxidation and makes it available to the cell. Even green plants use a process called photophosphorylation for converting the light energy it absorbs into chemical energy. This process is commonly known as photosynthesis.

Phosphorylation Reaction and Mechanism

Proteins often undergo a huge post-translational modification most of the times. Out of all the post-translational modification of proteins that happen, phosphorylation is the most important and is found almost everywhere. Of all the proteins that are available in the cell cytosol, 10% of them undergo phosphorylation. 

Phosphorylation reaction is one of the most widespread reactions that happen in human cells to phosphorylate the proteins that are present in the human proteome. The phosphorylation reaction that takes place in the cell is reversible in nature where catalysts such as kinases are used for the addition of the phosphoryl group and phosphatases catalyzes the removal of the phosphoryl group. 

In this reaction, ATP has the main function as it works as a phosphoryl donor or the phosphorylation reaction and acts as a reagent for hydrolysis of phosphoryl group in the dephosphorylation reaction. The entire reaction can be depicted as the hydrolysis reaction of ATP as the ΔG value is -12kcal / mol under cellular conditions and therefore, considered to be favourable for energy.

E + ATP → E―P + ADP, this is phosphorylation reaction 

E - P + H₂O →  P_i + E, this is dephosphorylation reaction

ATP + H₂O  →  P_i + ADP, this is the net result of above two reactions

From the above reactions, it is evident that phosphorylation is predominant in the post-translational modification that regulates protein functions in the body. Phosphorylation occurs at the end chain of three amino acids, tyrosine and threonine. The phosphate group (y-PO₃^2-) which is present at the terminal of universal phosphoryl group donor ATP, is attacked by a nucleophilic hydroxyl group (-OH) present in amino acid. This results in the transfer of the phosphate group to the amino side chain, and the entire reaction is facilitated by (Mg²⁺) ions. In order for the phosphoryl group to transfer easily to the nucleophilic hydroxyl group, magnesium ions bring down the threshold of phosphoryl transfer by chelating with γ- and 𝛽- phosphate. A large amount of free energy is released when the phosphate-phosphate bond in ATP is broken in order to form adenosine diphosphate ADP.

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Glucose and Glycolysis

Glycolysis is a very important process in which the glucose is broken down into two molecules of pyruvate in many steps with the help of various enzymes initiating the reaction at several stages. Precisely the process of glycolysis is carried out in ten steps and phosphorylation plays a major part in attaining the main end product. Phosphorylation initiates the first step of the preparatory stage, that is, half glycolysis and the last step of the payoff phase, that is, second glycolysis. As glucose is a very small molecule, thus, it has the ability to diffuse out through the membrane of the cell. Now when the phosphorylation of glucose happens in the first stage of glycolysis, glucose gets converted into glucose-6-phosphate which is relatively a bigger molecule than glucose. Therefore, it is trapped inside the membrane which then becomes negatively charged. This entire reaction is initiated by an enzyme called hexokinase. In the third phase of glycolysis, phosphorylation takes place and it converts fructose-6-phosphate into fructose-6- bisphosphate. This reaction is catalysed by phosphofructokinase. While the phosphorylation in the first step is initiated by ATP, phosphorylation in the payoff stage is maintained by inorganic phosphate.

Protein Phosphorylation

Protein phosphorylation is one of the most abundant and widespread post-translational modifications that occur in eukaryotes. Phosphorylation primarily occurs in the side chains of serine, threonine and tyrosine through phosphoester bond formation. Its occurrence is also evident in histidine, lysine and arginine through phosphoramidate bonds and in aspartic acid and glutamic acid through mixed anhydride linkage. Phosphorylation widely happens on human proteins at multiple non-canonical amino acids that include motifs that comprisethe histidine, aspartate, glutamate, arginine and lysine. Histidine phosphorylation takes place in both 1,3- N atoms of the imidazole ring. Being one of the most important PMT’s, protein phosphorylation plays a very important role in regulating cardiovascular, gastrointestinal, immunity, behavioural as well as actions of neurological irregularities. In addition to this, protein phosphorylation can contribute to one of the most critical pathological conditions, cancer. Studies found that the proteins in the human body guarded by the human genome are capable of undergoing protein phosphorylation.

Methods of Detection

Since phosphorylation has a huge impact on the human biological system and genomes and has the capability to fight against many diseases, a lot of methods have been developed to analyze the phosphorylation of the protein. One of the most common methods used to analyze the dynamics of phosphorylation of the entire protein family is the phosphoproteomic process. Though the small scale protein phosphorylation is generally performed to study small proteins, many modern methods are developed to analyse the dynamics of protein phosphorylation. Those methods are immunodetection, mass spectroscopy, phosphoprotein or phosphopeptide enrichment and kinase activity assays.