The cyclooxygenase isoforms are together with the most methodically studied and best comprehended mammalian oxygenases. Permitting the mode of action, pain management begin to take shape while the prostaglandin was concluded to be a major component in pain and inflammation in the mid 1930. After this became known, the mechanism for these inflammation mediators was determined in order to try to find a way to inhibit a series of prostaglandin. Acquiring two separate but linked active sites, the COXs catalyze the bis-dioxygenation and the following reduction of arachidonic acid (AA) to prostaglandin (PG) G2 and PGH2. In the biosynthetic pathway from arachidonic acid to prostaglandins, cyclooxygenase (COX) plays the role of central enzyme. This protein was distilled 20 years ago and above which was cloned in 1988. Years had passed another protein with an activity of COX was discovered and called COX-2. The proteins are greatly homologous in series and in a structure of three dimensions although the isoforms of COX are obtained from distinctive genes of different size and leads to distinct mRNA sequences. They have similar two catalytic sites, a COX site and a peroxidase, they also use the identical substrate and form a matching product. The comprehensive structures of the active COX sites are more or less similar to each other.
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However, there are very significant biological differences between COX-1 and COX-2. The last is a greatly inducible protein, absent from most tissues in normal conditions but increasing rapidly in response to inflammatory stimuli such as growth factors. Moreover, there are differences in substrate binding and, specifically, in inhibitor binding sites that allow the isoforms to be inhibited differentially. This difference is therapeutically significant and selective inhibitors of COX-2 displays anti-inflammatory potency without the gastric and renal toxicities of the aspirin-like drugs. Selective COX-2 inhibitors may also have vital effects on cell growth, development, or survival, reflecting the location of COX-2on the nuclear membrane of cells. (3)
There are two different active sites present in this enzyme. They are termed prostaglandin synthase. On one side, it has the cyclooxygenase active site while on the opposite side it has an entirely separate peroxidase site, which is needed to activate the heme groups that contribute in the cyclooxygenase reaction. COX-1 and COX-2 are homodimers of 576 and 581 amino acids, in that order.The enzyme complex is a dimer of identical subunits, so all in all, there are two cyclooxygenase active sites and two peroxidase active sites in close proximity. Each subunit has a knob covered with hydrophobic amino acids. These knobs anchor the complex to the membrane of the endoplasmic reticulum. The cyclooxygenase active site is buried deep within the protein, and is reachable by a tunnel that opens out in the middle of the knob. This acts like a funnel, guiding arachidonic acid out of the membrane and into the enzyme for processing as shown in Figure 1. (4)
Figure 1. Structure of Cyclooxygenase (5)
The COX active site is portrayed in yellow, the membrane binding domain motif showed in orange, catalytic domain in green and the peroxidase site appeared as red. As a group, these domains are made up of 25 alpha helices, seven 310 helices, four beta sheets as well as five disulfide bonds which help the interface binding of the two unique monomers in order to complete the enzyme. Then again, the fascinating crucial point of this enzyme is within a hydrophobic channel created by helices C and D which interact with the membrane as well as helices 6, 8, and 17 which lead to the cyclooxygenase active site of the enzyme as seen in Figure 2.
Figure 2. Hydrophobic channel of COX
Mechanism of Catalysis, Kinetics of Reaction and Mode of Regulation
The first step in the conversion of AA to the hydroperoxy-endoperoxide, PGG2, is abstraction of the pro-S hydrogen atom from carbon-13. The steps that follow are coherent with the mechanism of non-enzymatic lipid peroxidation, therefore the main contributions of COX to PGG2 formation are to limit the options for hydrogen abstraction. It is a requirement for cyclooxygenase catalysis that the enzyme must first be activated which is a process reliant on the peroxidase activity. The oxidation of the ferric heme to an oxo-ferryl porphyrin radical cation is led by two-electron reduction of a peroxide substrate. Tyrosyl radical in the cyclooxygenase active site is generated by the transfer of an electron to the heme from Tyr-385 of the protein. This radical is positioned perfectly to select the pro-S hydrogen from carbon-13 of AA, starting the cycloxygenase reaction. The final step of the reaction, reduction of the peroxyl radical to the hydroperoxide to form PGG2, regenerates the tyrosyl radical. Accordingly, activated COX can perform multiple turnovers without need to repeat the activation step. After initiating the cyclooxygenase reaction, the primary function of the peroxidase is to reduce the 15-hydroperoxy of PGG2 to the corresponding alcohol of PGH2 (1-3). (6)
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Figure 3. Mechanism of COX catalysis (7)
Kinetics of reaction deals with the rates of chemical processes. Figure 4 illustrates the pathway shows the inhibition of enzyme cyclooxygenase. The anti-inflammatory strength of the different NSAIDs were proportional to their action as cyclooxygenase inhibitors. It was also shown that cyclooxygenase inhibition created toxic side effects. (8)
COX-1, a systematic deviation between studied and fitted values was obvious at higher substrate concentrations. An inhibitory effect on Vmax was conducted which could not be effectively accounted for using a model for competitive inhibition. It was found that the observed data shows function of substrate concentration taken at various concentrations with either (a) COX-1 or (b) COX-2. (9)
The mode of regulation of COX shows that the regulation of COX-2 by prostanoids occurs at the transcriptional level. To be clearly established, it should remain the contribution of other prostanoid in the induction of COX-2. Intake of this leads to increases in levels of COX-2 protein. (10)
Figure 4. Cyclooxygenase Pathway (11)
Associated Diseases and Importance of COX to Human Health
Prostaglandins are widely spread in human tissues. They were found to be strong vasodilators and mediators when it comes to inflammation that can be seen in almost all tissues and organs. The encoded PGs by an early response gene also regulate other critical physiological responses not just playing a central role in inflammation. PGs are involved in diverse functions in humans, such as blood clotting, ovulation, bone metabolism, initiation of labor, nerve growth and development, blood vessel tone, wound healing, kidney function, and immune responses. On the other hand, there are 2 major aspects relevant to remember. Firstly, in the central nervous system, COX-2 is stated under normal conditions and plays a part in fundamental brain functions, such as synaptic activity, memory consolidation, and functional hyperemia. Secondly, ''neuroinï¬‚ammation'' is a much more organized reaction in peripheral tissues than inï¬‚ammation and is triggered and uphold by activation of resident cells, particularly microglia in many cases. (12)
Mostly of the associated diseases of COX belong to Inflammatory and Degenerative Brain Disease. One of these is the multiple sclerosis (MS), a disease which progressively affects the nerves of the brain and spinal cord. It typically starts in the early adult life and distinguished by perivascular infiltration of lymphocytes and macrophages into the brain parenchyma. With this, the expression of COX-2 and its involvement to the pathogenic events in MS have been explored in several animal models and in MS patients.In the development of the disease, the number of COX-2-positive endothelial cells increase in amount, mostly in areas of cellular penetration. The microglia-like cells known as macrophage expresses COX-1 spread throughout the brain parenchyma of control organism. (13)
Following is the Parkinson's disease which is the second most common neurodegenerative disorder, characterized by the loss of dopamine-containing neurons in the substantia nigra, which leads to severe movement disorders. Presently, there are different results on the ability of numerous NSAIDs to trim down neurodegeneration in cellular models. (14). Subsequently, Alzheimer's disease is relatively prominent among older people and is the most common form of dementia. Dementia is a disorder in brain the significantly affects a person's ability to perform daily activities.(15) They implies that several analyses of the two isomers of COX expressions have been conducted to AD brain tissues, providing a substantial yet still body of evidence pointing to the involvement of COX-2 resulting to neurodegeneration in AD. It was reported that as either decrease or increased of COX-2 mRNA levels in AD brains is possibly because of the short half-life of COX-2 individual variations (transcripts) of related inflammatory processes. Lastly, Creutzfeldt-Jakob disease (CJD). A disease which is rare and usually exhibits behavior changes, memory difficulties, vision problems and slow muscle coordination progress (ataxia), coma and then death. (16)
As COX have diseases associated which some are very uncommon, it also have the brighter side, COX as treatment. It also serves as inhibitor to compounds which are formed in response to inflammation that block cyclooxygenase enzymes, and by precancerous and cancerous tissues. The high-grade glioma stays one of the trickiest cancers to treat. A role of the ubiquitous enzyme, COX, specifically COX-2 was found out in cell proliferation and inhibition in control of cancer and apoptosis (cell death). (17)
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The study of the COX enzymes may be considered an advanced field coming from the perspective of the study of enzymes. There are a small number of enzymes of lipid biochemistry for which there is such an asset of structural and functional information.