What is Phosphorylation?
Phosphorylation is a chemical process in which a phosphoryl group (PO32-) 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 → Pi + E, this is dephosphorylation reaction
ATP + H2O → Pi + 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-PO32-) 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.
FAQs on Phosphorylation
1. What is phosphorylation in chemistry and biology?
Phosphorylation is a fundamental biochemical process involving the addition of a phosphoryl group (PO₃²⁻) to an organic molecule. This reaction is most often catalysed by enzymes called kinases. The primary donor of the phosphate group in biological systems is adenosine triphosphate (ATP). This process is crucial as it can increase the energy of a molecule or change its function, for example, converting glucose into glucose-6-phosphate to trap it inside a cell for metabolism.
2. What are the main types of phosphorylation for ATP synthesis in a cell?
There are three primary types of phosphorylation that lead to the synthesis of ATP:
- Substrate-Level Phosphorylation: This is the direct transfer of a phosphate group from a phosphorylated compound (the substrate) to ADP, forming ATP. This process occurs during glycolysis and the Krebs cycle.
- Oxidative Phosphorylation: This is the main source of ATP in aerobic organisms. It occurs in the mitochondria and involves the transfer of electrons through an electron transport chain, which powers the synthesis of ATP from ADP and inorganic phosphate (Pi).
- Photophosphorylation: This process is unique to photosynthetic organisms like plants and algae. It uses light energy to create a proton gradient across the thylakoid membrane in chloroplasts, driving ATP synthesis.
3. What is the specific role of phosphorylation in the process of glycolysis?
Phosphorylation plays two critical roles in glycolysis. First, in the preparatory phase, glucose is phosphorylated to form glucose-6-phosphate. This step, catalysed by the enzyme hexokinase, uses one ATP molecule and serves to trap glucose inside the cell. Second, fructose-6-phosphate is phosphorylated to become fructose-1,6-bisphosphate, a key regulatory and irreversible step that commits the molecule to the glycolytic pathway.
4. What is the difference between phosphorylation and dephosphorylation?
Phosphorylation and dephosphorylation are opposing processes that regulate cellular activities. Phosphorylation is the addition of a phosphate group to a molecule, typically catalysed by enzymes called kinases, which usually activates a protein. In contrast, dephosphorylation is the removal of a phosphate group, a reaction catalysed by enzymes called phosphatases, which often deactivates the protein. This on/off cycle is essential for cell signalling and metabolic control.
5. How does the phosphorylation of proteins serve as a regulatory mechanism?
Protein phosphorylation is a major type of post-translational modification that acts as a molecular switch. When a kinase adds a negatively charged, bulky phosphate group to specific amino acids (like serine, threonine, or tyrosine), it can change the protein's three-dimensional shape (conformation). This change alters the protein's activity, its ability to bind to other molecules, or its location within the cell, thereby turning its function on or off.
6. Why is ATP known as the universal phosphoryl group donor in most biological reactions?
ATP is the universal donor because of the high-energy phosphoanhydride bonds linking its three phosphate groups. The hydrolysis of these bonds, particularly the terminal one, releases a significant amount of free energy (ΔG). This energy release makes the transfer of the phosphate group to another molecule an energetically favourable process, allowing it to drive otherwise non-spontaneous reactions forward, which is essential for metabolism and cell function.
7. How does phosphorylation act as a molecular 'on/off switch' to control protein function?
Phosphorylation acts as a switch by causing a significant change in a protein's properties. The addition of a phosphate group by a kinase introduces a bulky, negative charge. This can attract positively charged amino acids or repel negatively charged ones, forcing the protein to change its shape. This new shape might expose an active site (turning it 'on') or hide it (turning it 'off'). The process is reversible by phosphatases, allowing for rapid and precise control over cellular processes.
8. Besides its role in glycolysis, what are some other critical cellular processes regulated by phosphorylation?
Phosphorylation is a ubiquitous regulatory mechanism that controls a vast range of cellular activities beyond glycolysis. Key examples include:
- Signal Transduction: Transmitting external signals from the cell membrane to the nucleus to alter gene expression.
- Cell Cycle Control: Regulating the progression of a cell through different phases like interphase and mitosis via cyclin-dependent kinases (CDKs).
- Muscle Contraction: Controlling the interaction between actin and myosin filaments.
- DNA Repair: Activating proteins that detect and repair damage to the cell's DNA.
9. Can phosphorylation occur on any amino acid in a protein? Explain the reason.
No, phosphorylation does not occur on all amino acids. In eukaryotes, it primarily happens on amino acids that have a hydroxyl (-OH) group in their side chain. The oxygen atom in the -OH group acts as a nucleophile to attack the phosphate group of ATP. The three main target amino acids are Serine (Ser), Threonine (Thr), and Tyrosine (Tyr), which account for the vast majority of protein phosphorylation events.
10. What is the key difference in purpose between photophosphorylation in plants and oxidative phosphorylation in animals?
The fundamental difference lies in their energy source and purpose. Photophosphorylation in plants captures light energy and converts it into chemical energy in the form of ATP within chloroplasts. This ATP is then immediately used to power the Calvin cycle for carbon fixation (making sugars). In contrast, oxidative phosphorylation in animals releases chemical energy stored in food molecules (like glucose) to produce ATP in mitochondria, which then powers all other general cellular activities.
