Structural Biochemistry/Enzyme Regulation/Phosphorylation
Phosphorylation
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Phosphorylation is an effective way of regulating proteins. About 30% of proteins in eukaryotic cells are phosphorylated. The enzymes that are responsible for these reactions are known as protein kinases. There are about 100 homologous protein kinases in yeast and 500 in human beings. When ATP is hydrolyzed in a test tube, the release of free energy merely heats the surrounding water. In an organism, this same generation of heat can sometimes be beneficial. For instance, the process of shivering uses ATP hydrolysis during muscle contraction to generate heat and warm the body. In most cases in the cell, however, the generation of heat alone would be an inefficient use of a valuable energy resource. Instead, with the help of specific enzymes, the cell is able to couple the energy of ATP hydrolysis directly to endergonic processes by transferring a phosphate group from ATP to some other molecule, such as the reactant. The recipient of the phosphate group is then said to be phosphorylated. The key to coupling exergonic and endergonic reactions is the formation of this phosphorylated intermediate, which is more reactive than the original unphosphorylated molecule.
ATP must be present for phosphorylation because it is needed as a donor. One of the phosphoryl groups of ATP is transferred to a specific amino acid. The acceptor is one of three amino acids with a hydroxyl group as a side chain: serine, threonine, and tyrosine. Tyrosine is handled by a different protein kinase than the other two. The reaction below shows how ATP donates one of the phosphoryl groups to a phosphorylated protein.
Protein phosphatases reverse the effects of kinases by catalyzing the removal of the phosphoryl group attached to proteins. The enzyme hydrolyzes and breaks the bond attaching the phosphoryl group.
It is important to note that phosphorylation and dephosphorylation reactions are not the reverse of one another. The former takes place through the action of protein kinase and ATP cleavage, whereas the latter will only take place in the presence of a phosphatase.
Reasons for Effectiveness
edit1) A phosphoryl group adds two negative charges to the protein. These changes alter substrate binding and catalytic activity.
2) A phosphoryl group can form 3 or more hydrogen bonds and can be tetrahedral.
3) The free energy of phosphorylation is very large. Therefore it can change the equilibrium by a large factor.
4) Phosphorylation and dephosphorylation take place in less than a second or over a span of hours.
5) Phosphorylation produces highly amplified effects. One kinase changes hundreds of target proteins in a short interval.
6) Phosphorylation is irreversible.
7) ATP is the energy needed, and thus it links the process to bio energetics.
Phosphorylation is important to the regulation of cells by regulating insulin, water balance, and homeostasis of the cell. The enzyme GSK-3 by AKT (Protein kinase B) regulates the insulin pathway. Na+/K+ ATPase regulate water balance and homeostasis of the cell.
Protein Kinase A
editProtein kinase A (PKA) is an enzyme that is regulated by cyclic AMP (cAMP). This is common in the "flight or fight response". The hormone Epinephrine signals the synthesis for Cyclic adenosine monophosphate which subsequently activates protein kinase A. The kinase regulates target proteins through phosphorylation of serine and threonine.
PKA is not activated until cAMP binds to the regulatory subunit. This stops inhibition of PKA. The complex (R2C2) has a pseudosubstrate sequence of R that occupies the active site of PKA. When cAMP binds, the R chains move so it is no longer inhibiting the active site. The R chain has the sequence Arg- Arg- Gly- Ala- Ile.
The figure above shows how once cAMP is bound to the binding sites, the pseudosequence is no longer blocking the active site of PKA. This is how PKA is regulated.
The protein kinase A holoenzyme is a heterotetramer made up of two types of subunits: 1)A catalytic subunit which contains the enzyme's active site. It also contains a domain that binds ATP (the source of phosphate) and a domain that binds the regulatory subunit. 2)A regulatory subunit which consists of two molecules of this subunit bind one another in an anti-parallel orientation to form a homodimer; for type I subunits, this binding is covalent through disulfide bonds. This subunit also has two domains that bind cyclic AMP, a domain that interacts with a catalytic subunit, and a inhibitory domain that serves as a substrate or pseudosubstrate for the catalytic subunit. Regulatory subunits can also modulate catalytic subunit activity.
Regulating Activity: Intracellular concentration of cyclic AMP provides a very simple control mechanism over activity of protein kinase A.
At low cyclic AMP levels, catalytic subunits are bound to a regulatory subunit dimer and are inactive. As the concentration of cAMP increases to ~10nM and above it binds to the regulatory subunits, which gives way to an allosteric change in conformation which causes unleashing of the catalytic subunits. Free catalytic subunits are active and begin to phosphorylate their targets.
Glycogen phosphorylase
editGlycogen is the polysarccharide of glucose. It serves as short term energy storage in animal cells. Glucose is a monosaccharide, an important carbohydrate in biology. It is used by the cell as a source of energy and as an metabolic intermediate.
Phosphorylase catalyzes the interconversion of glycogen and glucose-1-phosphate by the reaction,
Glycogen + Pi --> Glycogen + glucose-1-phosphate
where glucose-1-phosphate is then converted to glucose-6-phosphate by phosphoglucomutase, since the glucose-1-phosphate isomer cannot be metabolize easily, whereas the glucose-6-phosphate acts as fuel for glycolysis and pentose phosphate pathway.
Phosphorylase activity is regulated by reversible phosphorylation. Phosphorylase a is the active form, while phosphorylase b is inactive.
Metabolic Disorders Caused by Defective Enzymes Controlling Phosphorylation
editAn example of a metabolic disease caused by defective enzymes is Lafora disease. Lafora disease, named after Dr. Gonzalo Lafora, is a metabolic disorder that is caused by defective enzymes that controls phosphorylation. It is a neurodegenerative disease caused by insoluble glucan accumulating in the cytoplasm. Glucan is a type of complex carbohydrate that is made up of glucose linked together by glycosidic bonds. Lafora disease not only causes epilepsy, but also progressive central nervous system degenerations. This ultimately results in the death of the patient. It is thought that the compilation of cytoplasmic lipid bodies (LB) trigger neuronal cell death and seizures.
It is recognized that in almost half of Lafora disease cases, the EPM2A (epilepsy, progressive myoclonus 2A) gene is mutated. The EPM2A gene encodes the bimodular protein called laforin. Laforin is associated with regulating glycogen metabolism. Glycogen stores long-term energy. Furthermore, about 20% if Lafora disease cases are a result of mutations in EPM2B (epilepsy, progressive myoclonus 2B), which encodes the protein called malin. Malin is associated with the binding, ubiquitylating, and promoting the degradation of laforin.
It is determined that insoluble glucan accumulating in the cytoplasm causes Lafora disease, although the molecular cause of this disease is still unknown. This is due to the fact that the function of laforin is obscure because its substrate is still unidentified. There are various hypotheses, but one prominent hypothesis is that laforin dephosphorylates glycogen molecules as they are synthesized. Without laforin, glycogen metabolism results in a slightly more phosphorylated glucan and would eventually lead to a Lafora body. Another hypothesis is that laforin recruits malin to the site of glycogen synthesis. Malin regulates the synthesis of glycogen by ubiquitylating protein targeting to glycogen (PTG), glycogen synthase (GS), glycogen debranching enzyme (GDE), and laforin to inhibit LB formation.