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# Phosphorylation

Proteins are one of the essential nutrients of human body as it acts as building block of body tissue. Although it provides less amount of energy compare to carbohydrates, yet it can act as fuel source for body in necessary conditions.

Proteins are composed of amino acids which bonded together through peptide linkage and form polypeptide chains. These polypeptide chains are arranged in a specific manner to form proteins.

The structure of proteins can be defined as four types.

• Primary structure: Amino acids act as monomer units for proteins. They bonded through peptide linkage and form polypeptide chain. The sequence of amino acids in polypeptide chain is known as primary structure of proteins. The formation of peptide bonds is an enzyme catalyzed condensation reaction and completed through loss of water.
• Secondary structure: The folding of polypeptide chains forms secondary structure of proteins. There are two common secondary structures of proteins known as alpha helix and beta pleated sheets which held together by hydrogen bonds and provide a proper shape and stability to molecule.
• Tertiary structure: The three dimensional structure of protein which held together through various interactions like disulphide bonds, ionic bond, hydrogen bond hydrophobic and hydrophilic interactions are known as tertiary structure. Tertiary structure of proteins can be mainly two types:
1. Globular: It’s a ball-like structure where hydrophobic parts oriented towards the centre and hydrophilic are towards the edges. For example; enzymes, plasma proteins, antibodies etc.
2. Fibrous: These are long fibers forms which consist of repeated sequences of amino acids. These type of structures found in water insoluble proteins like collagen.
• Quaternary structure: Some polypeptide chains consist of some inorganic components called as prosthetic group. This type of protein structure is known as quaternary structure.

## Define Phosphorylation

As we know that proteins are composed of amino acids and each amino acid has a specific structure. All amino acids have one carboxy group, one amino group, one hydrogen and one side chain.
1. This side chain is vary in each amino acid and responsible for characteristic properties of amino acid.
2. The addition of any other group on amino acid can affect structure as well as reactivity of proteins.
3. The addition of a phosphate to one of the amino acid side chains of a protein is known as phosphorylation.
4. The addition of phosphate group on amino acid affects the orientation of protein structure as the phosphate group is a negatively charged group (PO43-).

Phosphorylation process is a reversible process and proteins can back to original conformation after removing the phosphate group. The removal of phosphate group is known as dephosphorylation process. Phosphorylation process is an enzyme catalyzed process and enzyme involves in process are called as protein kinases.

Protein phosphatases enzyme is involved in dephosphorylation process. In general the hydroxyl group of amino acid like serine, threonine or histidine is most common target for phosphorylation. The phosphate group is carrying by energy molecules like ATP and place it on the amino acid side chain in protein.

## Protein Phosphorylation

The phosphorylation of proteins through serine, threonine or tyrosine residue in the presence of protein kinase is known as protein phosphorylation. It is a reversible post translational modification process which regulates protein functions and also called as phospho regulation. Protein phosphorylation is a rapid process taking a few seconds and does not require new protein to be made or degraded.
1. Protein phosphorylation is a reversible process and occurs with many enzymes and receptors.
2. It activates and deactivates the functions of enzymes and receptors. It is a regulatory mechanism which can occur in both prokaryotic and eukaryotic organism and occurs on the serine, threonine and histidine residues.
3. In eukaryotic, histidine phosphorylation is much common while in prokaryotic proteins phosphorylation occurs on the serine, threonine, tyrosine, histidine or arginine or lysine residues.
4. The phosphorylation of protein change the conformation of molecule as the addition of a phosphate molecule to a polar R group of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic portion of molecule.

Phosphorylation also regulates cell signaling in response to a wide range of external and internal stimuli in plants. The addition of phosphate group modulates the protein functions due to addition of amino acid residue like serine, threonine and some time for tyrosine also.

## Phosphorylation Reaction

There are a huge number of covalent post translational modifications in proteins like fatty acid acylation, acetylation, phosphorylation and glycosylation. Out of these modifications, phosphorylation is the most common post translational modifications which occur in cytosol.It's a reversible enzymatic process which involves kinase and phosphatase enzymes in process with ATP as phosphoryl donor. It's an energetically favorable process in cellular condition with Î”G -12 kcal/mol. The overall reaction can be represented as below.
Phosphorylation: E +ATP $\to$ E-P +ADP

Dephosphorylation: E-P + H2O $\to$ E + Pi
Net reaction: ATP + H2O $\to$ADP + Pi

The addition of phosphoryl group added two negative charges on protein which modify and disrupt the electrostatic interactions of protein. The change in conformation of protein affects the substrate binding and catalytic activity of phosphorylated enzyme.

## Substrate Level Phosphorylation

The transfer of phosphate group from substrate to ADP to form ATP is known as substrate level phosphorylation. It's an enzymatic process for the formation of ATP. Hence the production ATP from ADP by a direct transfer of a high-energy phosphate group from a phosphorylated intermediate metabolic compound in an exergonic catabolic pathway.

Some common examples of substrate level phosphorylation are as follow.
2. In Citric Acid Cycle GDP+Pi forms GTP by simple conversion forms ATP.
3. Conversion of phosphoenolpyruvate to pyruvate
4. Conversion of glucose to phosphoenolpyruvate during glycosylation.

## Oxidative Phosphorylation

Oxidative phosphorylation is synthesis of ATP molecules using energy released by oxidation of reduced co-enzymes namely NADH and FADH2. These co-enzymes are produced during respiration. Oxidative phosphorylation is the final metabolic pathway of cellular respiration preceded by glycolysis and citric acid cycle. Oxidative phosphorylation is carried out by ATP synthase, the fifth complex of electron transport chain. ATP synthase is present in F1 particles of the complex.
• These particles are found in inner mitochondrial membrane. Electron transport through other four complexes of electron transport chain generates a proton gradient with higher proton concentration on F0 side as compared to that of F1 side.
• Energy liberated during transfer of electron from one carrier to other pushes protons to outer side of inner mitochondrial membrane.
• Transport of electrons from NADH and FADH2 over electron transport chain pushes three and two pairs of protons respectively to outer side.
• Transport of protons down their concentration gradient from outer side to inner side of inner mitochondrial membrane over F0 particles activates F1 particles to serve as ATP synthase.
• Energy of proton gradient is used for synthesis of ATP from ADP and inorganic phosphate.
• Oxidation of one molecules of NADH and FADH2 each produce three and two ATP molecules respectively.

## Cyclic Phosphorylation

Process of ATP synthesis using sunlight is known as photophosphorylation. Energy from sunlight creates a proton gradient. Although process of proton gradient synthesis resembles that of the electron transport chain of respiration, yet owing to its light-dependency, the process is called Photophosphorylation. Photosynthetic electron transport and splitting of water molecule result in accumulation of protons in thylakoid space. The increased proton concentration in thylakoid space results in development of proton gradient across the thylakoid membrane. Movement of protons down the concentration gradient through ATP synthase complex drives ATP synthesis from ADP and inorganic phosphate.

Photophosphorylation operates through cyclic and non-cyclic processes.
Cyclic photophosphorylation is the process where an electron, expelled by excited photocentre, is returned to its source photocentre after passing through a series of electron carriers.Cyclic photophosphorylation involves photosystem I only. It occurs under low sunlight that does not favor CO2 fixation and hence does not poses a need for formation of NADPH.

The photocentre P700 is oxidized after absorbing light energy. The expelled electron is passed through a series of electron carriers namely A0 (a special P700 chlorophyll molecule), a quinone, FeS complex, ferredoxin, plastoquinone, cytochrome b-f complex and plastocyanin. Plastocyanin return the electron to the P700. During electron transfer, cytochrome complex serves as site for development of proton gradient that finally drives synthesis of ATP from ADP and inorganic phosphate.

## Non-Cyclic Photophosphorylation

Non cyclic photophosphorylation involves both photosystem I and II. It does not require returning of expelled electron to its source photocentre. Light driven splitting of water results in release of electron that is picked by photocentre of PS II (P680). Absorption of light energy by P680 results in release of this electron to be passed through a series of electron carriers (pheaophytin, PQ, cytochrome b-f complex and plastocyanin).

Again cytochrome complex serves as site for development of proton gradient that finally drives synthesis of ATP from ADP and inorganic phosphate. Plastocyanin passes the electron to photocentre of PSI (P700 ). The electron is passes to A0 (a special P700 chlorophyll molecule), a quinone, FeS complex, ferredoxin and finally to NADP+. The NADP+ is reduced via NADP-reductase to form NADPH.

## Histone Phosphorylation

Histone proteins are involved in packaging of eukaryotic DNA in nucleosomes. Histone phosphorylation is the key process in many of important biological processes namely transcription, DNA repair, programmed cell death and chromatin condensation and decondensation. It has been shown that phosphorylation of serine 10 and 28 in eukaryotic histone H3 is related to gene activation in mammalian cells. Serine phosphorylation results in increased HAT activity, thus causing transcriptional activation of genes. Altered patterns of histone phosphorylation impart better understanding of many diseases as well as serve in development of protein kinase targeted drugs.