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An enzyme is a complex protein molecule that catalyzes a specific biochemical reaction. Enzymes action is accomplished by lowering of activation energy of a reaction, thus allowing the reaction to proceed at its steady state. Enzyme could be recovered unaltered in the end of the reaction and therefore could be used further. Enzymes possess high degree of specificity for substrates. They accelerate the specific biochemical reactions and act in aqueous physiological medium under mild temperature and cellular pH conditions. The enzymes study provides better understanding of cell survival and proliferation. Enzyme catalysis lead to hundreds of reactions in metabolic pathways by which nutrient molecules are degraded, chemical energy is generated and biological macromolecules are synthesized from simple precursors. There are around 3000 known enzymes and probably several hundreds more are still undiscovered (Murray, 1996).
Mechanism of Enzyme Action
In order for a reaction to proceed, reactant molecules must possess sufficient energy to cross a potential energy barrier, the activation energy. All molecules contain varying amounts of energy depending, for example, on their recent collision history but, generally, only a few have sufficient energy for reaction. The lower the potential energy barrier to reaction, the more reactants would have sufficient energy to undergo reaction and, hence, the faster the reaction will occur. Enzyme catalysis, occur by forming a transition state, with the reactants, of lower free energy than would be found in the uncatalysed reaction. Even modest reductions in this potential energy barrier may produce large increases in the rate of reaction (e.g. the activation energy for the uncatalysed breakdown of hydrogen peroxide to oxygen and water is 76 kJ M-1 whereas, in the presence of the enzyme catalase, this is reduced to 30 kJ M-1 and the rate of reaction is increased by a factor of 108, sufficient to convert a reaction time measured in years into one measured in seconds).
The basic mechanism by which enzymes catalyze chemical reactions begins with the binding of the substrate or substrates to the active site of the enzyme, which causes changes in the distribution of electrons in the chemical bonds of the substrate and ultimately facilitates the reactions that lead to the formation of products. The products are released from the enzyme surface to regenerate the enzyme for another reaction cycle.
A schematic diagram showing the free energy profile of the course of an enzyme catalysed reaction involving the formation of enzyme-substrate (ES) and enzyme-product (EP) complexes is as follows
The active site of the enzyme has a unique geometric shape that is complementary to the geometric shape of a substrate molecule, similar to the fit of puzzle pieces. This means that enzymes specifically react with one or very few compounds of similar structures. The high specificity and efficiency of enzymes can be explained by the manner they associate with the substrate.
Theories of enzyme binding
There are basically two main theories available to explain the enzyme and substrate binding
Key & lock theory
Induced fit theory
KEY AND LOCK THEORY
The first theory that helps to explain phenomenally efficient catalytic efficiency of enzymes is called lock and key model. According to this model, each enzyme molecule may have one active site on the surface of the enzyme molecule itself. Whereby, a substrate is attached to the enzyme to form enzyme-substrate complex. The polar and non-polar groups of the active site attract compatible groups on the substrate molecule so that the substrate molecule can effectively lock into the cavity, and position itself for the necessary collisions i.e., bond breaking and formations that must take place for successful conversion to a product molecule. Once the product molecule is formed, the electrical attractions that made the substrate molecule adhere to the active site no longer operate and the product molecule can disengage itself from the active site thus freeing the site, for another incoming substrate molecule. This process occurs in a highly efficient manner, hundreds or even thousands of times in a short time span (Murray, 1996).
INDUCED FIT THEORY
All the chemical pathways of reactions could not be adequately explained by using the so-called rigid enzyme model assumed by the lock and key theory. Therefore, a modified model called the induced-fit theory has been proposed.
The induced-fit theory assumes that the substrate plays a role in determining the final shape of the enzyme, which is partially flexible. This explains phenomenon of substrate binding to the enzyme but no reaction takes place, because the enzyme has been distorted too much. Other molecules may be too small to induce the proper alignment and therefore cannot react. Thus, only the proper substrate is capable of inducing the proper alignment of the active site.
The substrate is represented by the magenta molecule; the enzyme protein is represented by the green and cyan colors. The cyan colored protein is used to more sharply define the active site. The protein chains are flexible and fit around the substrate
The favored model for sthe enzyme-substrate interaction is the induced fit model. This model also suggests that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen bindings.
Types of Enzyme Inhibitors
Enzyme inhibitors are molecules or ions that interact in some way with the enzyme to prevent it from working in the normal manner. Depending upon their mode of inhibition, they have been classified into following major categories.
Inhibitors that bind irreversibly and covalently with the enzyme. Enzyme inhibitor complex is highly stable not dissociate. e.g., Diisopropylfluorophosphate is an irreversible protease inhibitor. The enzyme hydrolyses the phosphorus-fluorine bond, but the phosphate residue remains bound to the serine in the active site, deactivating it (Murray, 1996).
E + I ï‚® EI complex
Reversible inhibitors are molecules or ions which bind reversibly and noncovalently. Reversible inhibitors are divided into following major type's competitive, noncompetitive, uncompetitive and mixed type of inhibition (Murray, 1996).
A competitive inhibitor is a compound, molecule or ion which closely resembles in the chemical structure and molecular geometry of the physiological substrate. The inhibitor competes with the substrate for the same active site as the substrate molecule. The inhibitor adheres to the enzyme and prevents any substrate molecules from reaching to the active site. However, a competitive inhibition is usually reversible if sufficient substrate molecules are available to ultimately displace the inhibitor. Therefore, the degree of enzyme inhibition depends upon [I], [S], Km, Ki, the inhibitor and the substrate concentrations (Murray, 1996).
E + S â†” ES â†” E +P
E + I â†” EI
B. Noncompetitive Inhibitors
A classical noncompetitive inhibitor has no effect on substrate binding site. S and I bind reversibly, randomly, and independently at different binding sites of the enzyme. This means that I can bind to E and ES complex and S can bind to E and El complex and the resulting ESI complex is catalytically inactive. Inhibitor might prevent the proper positioning of the catalytic center (Murray, 1996).
E + S â†” ES ï‚® E + P
ES + I â†” ESI
EI + S â†” EIS
C. Uncompetitive Inhibitors
A classical uncompetitive inhibitor is a compound that binds reversibly to the enzyme-substrate complex yielding an ESI complex. The inhibitor does not bind to the free enzyme (Murray, 1996).
E + S â†” ES ï‚® E + P
ES + I â†” ESI
D. Mixed type Inhibitors
Mixed type inhibitors fall into the category of noncompetitive type of inhibitors. These can also results from an inhibitor's binding at two different mutually exclusive sites. Binding at one site completely excludes the substrate, while binding of inhibitor at the second site has no effect on the binding of substrate but the resulting ESI complex is catalytically inactive (Murray, 1996).
E. Product Inhibition and Feedback Inhibition
When immediate P of an enzyme catalyzed reaction is accumulated and thi excess of P may lower the enzymatic reaction by occupying the active site of the enzyme, the type of inhibition is called product inhibition. However, if the endproducts of a pathway inhibit the activty of any one of the enzymes of the pathway, it is called feedback inhibition.
Factors Affecting Enzyme Activity
Activity of enzymes is affected by several factors which can increase or decrease the rate of reaction. These factors include the type and concentration of the buffer, pH of the assay medium, temperature, concentration of enzyme and concentration of the substrate, coenzyme, and cofactors. Various types of inhibitors and activators and allosteric modulators also affect the enzyme activity. Several types of covalent modifications like phosphorylation/dephosphorylation, glycosylation, biotinylation, hydroxylation etc also have marked effect on the velocity of the reaction (Murray, 1996).
High Throughput Screening and Drug Discovery
HTS is the screening of large number substances in an efficient and timely manner. The ultimate goal is the discovery of active substances which can serve directly or after optimization as templates for molecules of commercial value. The market mostly is the pharmaceutical sector but other life science area is also employing HTS as a part of their drug discovery (Kidlington, 2007).
The method of molecular biology can achieve the modification, over expression and purification of most drug targets and have opened the possibility of screening approaches on single isolated biochemical targets. The availability of potentially modified and enzymes stimulated the development of a plethora of novel assay technologies. Typically development of HTS assay techniques tends toward a simplification of steps i.e., the favored goal is an add, mix and read process. In its simplest form assay reagents are mixed with the compounds to be tested and after short incubation the activity is measured in some form of physically detectable signals such signal in most cases optical in nature i.e., a change of optical density fluorescence, or emission of light. For better signal-to-background resolution such readout signal can be further modulated by filtering of appropriate wavelength or can be time resolved (Kidlington, 2007).
Enzymes of Study
Acetyl and Butyryl cholinesterase Enzyme
AChE EC 188.8.131.52 comprise a family of enzymes which include serine hydrolases. They share about 55% of amino acid sequence identity, and have similar catalytic properties. The different specificities for substrates and inhibitors are due to the difference in amino acid residues of the active sites of AChE and BChE The enzyme system is responsible for the termination acetylcholine at cholinergic synapses (Cyglar et al., 1993).
The major function of AChE is to catalyze the hydrolysis of the neurotransmitter acetylcholine and termination of the nerve impulse in cholinergic synapses (Quinn, 1987).
Acetyl CoA + Choline =========> Acetylcholine + CoA
Acetylcholine <==========> Choline + acetate
H1 and H2 receptor antagonists have been shown to possess AChE inhibitory activities It has been found that BChE is present in significantly higher quantities in Alzheimer's plaques than in the normal age related non dementia of brains. Hence the search for the new cholinesterase inhibitors is considered to be an important and ongoing strategy to introduce new drug candidates for the treatment of AD and other related diseases (Bertaccini, 1982).
Acetyl cholinesterase and Its Inhibitors
A variety of neurological and neuromuscular disorders involve a diminution of cholinergic activity. Often the most effective treatments are ligands which inhibit the breakdown of acetylcholine. Acetyl cholinesterase inhibitors have been used clinically in the treatment of myasthenia gravis, a degenerative neuromuscular disorder, glaucoma and more recently Alzheimer's disease. In addition, cholinesterase inhibitors are widely utilized as pesticides and, if misused, can produce toxic responses in mammals and man.
Acetyl cholinesterase is a tetrameric protein which catalyzes the hydrolysis of acetylcholine. The active site of AChE includes a serine hydroxyl group, which is rendered more nucleophilic through the proton-acceptor action of a nearby histidine residue. The serine residue exerts a nucleophilic attack on the carbonyl carbon of ACh. A tetrahedral transition state is reached, which results in serine acetylation and the loss of free Choline. The acetyl group binds to histidine as an N-acetate, but is hydrolyzed rapidly to yield free Choline, acetate, and the free enzyme (Foye et al., 1995).
Some inhibitors of acetyl cholinesterase act by competitively blocking hydrolysis, without reacting with the enzyme. Others inhibit by acylating the serine hydroxyl group, forming a carbamyl ester, which is more stable than acetate and less likely to leave the active site. The competitive blocker edrophonium is a quaternary compound that blocks the enzyme by binding to the active site (Foye et al., 1995).
Clinical Importance of AChE inhibitors
The alkaloids physostigmine and neostigmine act as metabolic inhibitors of acetyl cholinesterase. The carbamyl ester formed by these compounds is much more stable than acetate half-life measured in minutes as opposed to microseconds. The cholinesterase inhibitors are widely used to treat glaucoma a disorder characterized by increased intraocular pressure. Acetylcholine reduces intraocular pressure, and cholinesterase inhibitors such as physostigmine are useful in treating the disease. Another major use of cholinesterase inhibitors is for treatment of myasthenia gravis, an autoimmune disease in which antibodies are formed against the nicotinic receptor at the neuromuscular junction. The antibodies bind to nicotinic receptors to cause a profound muscle weakness and paralysis. Cholinesterase inhibitors can alleviate the symptoms of myasthenia by increasing muscle strength and endurance (Foye et al., 1995).
Alzheimer's disease (AD) is a neurodegenerative disorder that currently affects nearly 20% of the population in industrialized countries. The risk of (AD) increases in the individuals beyond the age of 70 and it is predicted that the incidence of (AD) will increase threefold within the next fifty years with an increase in aging population. Brain regions involved in learning and memory process, including the temporal and frontal lobes, are reduced in size in (AD) patients, as a result of degeneration of synapses and death of neurons. Definitive diagnosis of (AD) requires post mortem examination of the brain, which must contain sufficient number of plaques and tangles to qualify as affected by (AD) (Dickson, 1997).
The deficiency in cholinergic neurotransmission in (AD) has led to the development of cholinesterase inhibitors as the first line of medicines for the symptomatic treatment of this disease. The clinical benefits of these agents include improvements in stabilization, cognition function and behavior. The common mechanism of action involves in this class of agents is an increase in available acetylcholine through inhibition of the AChE. There is substantial evidence that the cholinesterase inhibitors, such as donepezil, galanthamine and rivastigmine, decrease AChE activity in a number of brain regions in patients with (AD).
Lipoxygenases (EC 184.108.40.206, LOX) are members of a class of non-heme iron-containing dioxygenases that catalyze the addition of molecular oxygen to fatty acids containing a cis, cis-1,4-pentadiene system to give an unsaturated fatty acid hydro peroxides. It has been found that these LOX products play a key role in variety of disorders such as bronchial asthma, inflammation (Steinhilber, 1999) and tumor angiogenesis (Nie et al., 2002).
Sources of Lipoxygenase
First LOX enzyme was discovered from soybean (Andre et al., 1932). LOX enzyme is distributed throughout the plant kingdom (Gardner, 1991). It has been isolated and purified from cucumber (Philips et al., 1978), tea chloroplast (Hatanaka et al., 1979), wheat (Wallance et al., 1975), potatoes (Galliard et al., 1980) and rice (Yamamto et al., 1980). LOX are found in human, mouse, rat, pig and rabbit. In human tissues LOX is expressed in platelet, eosinophils, synovial fluid, neutrophils, colonic tissues, lung tissues, monocytes and bone marrow cells (Steinhilber et al., 1999).
Mechanism of Action of Lipoxygenase
Arachidonic acid (AA) is converted to a variety of linear hydroperoxy acids by separate pathways involving a family of lipoxygenases. Neutrophils contain 5-LOX which converts the arachidonic acid to 5-hydroxy-6, 8, 11, 14-eicosatetraenoic acid (5-HPETE). 5-HPETE is converted into a series of leukotrienes and the nature of the final product varies according to the tissue. LOX enzymes are not affected by NSAIDS. Leukotrienes are mediators of allergic response and inflammation. Inhibitors of 5-LOX and leukotrienes receptor antagonist are used in the treatment of asthma (Tomchick et al., 2001). Details are given in Figure 1 and Tables 3-5.
Figure 1. Biosynthesis of Leukotrienes via HPETEs
LOX creates hydro peroxides from polyunsaturated fatty acids by an insertion of molecular oxygen. Both arachidonic acidÂ (omegaâˆ’6) and eicosapentoic acidÂ (omegaâˆ’3) can be acted on by LOX enzyme, resulting in leukotrienes.Â LOX produces seriesÂ 4 leukotrienes from arachidonic acid or seriesÂ 5 leukotrienes from eicosapentoic acid. (http://www.benbest.com/health/essfat.html)
Table 3. Biochemical Classification of Lipoxygenases
linoleate: oxygen 13-oxidoreductase
linoleate + O2 = (9Z,11E,13S)-13-hydroperoxyoctadeca-9,11-dienoate
arachidonate: oxygen 12-oxidoreductase)
arachidonate + O2 = (5Z,8Z,10E,12S,14Z)-12-hydroperoxyicosa-5,8,10,14-tetraenoate
(arachidonate: oxygen 15-oxidoreductase)
arachidonate + O2 = (5Z,8Z,11Z,13E,15S)-15-hydroperoxyicosa-5,8,11,13-tetraenoate
(arachidonate: oxygen 5-oxidoreductase)
arachidonate + O2 = leukotriene A4 + H2
(arachidonate: oxygen 8-oxidoreductase)
arachidonate + O2 = (5Z,8R,9E,11Z,14Z)-8-hydroperoxyicosa-5,9,11,14-tetraenoate
Compounds that combine with the enzyme and prevent the formation of enzyme-substrate complex are the lipoxygenase inhibitors. Lipoxygenase-arachidonic acid complex formation results in the formation of the eicosanoid products like hydroxyeicosatetraenoic acid (HETEA) and various leukotrienes. Lipoxygenase inhibitors are classified into five main categories according to their mechanism of inhibition (Nelson and Seitz, 1994).
Catechols and related substances
Hydroxamic acid derivatives
LOX activating protein inhibitors
A majority of LOX inhibitors belong to the group of antioxidants. These substances are mostly one electron reducing agents, competitively occupying active center of the enzyme and reduce the active ferric ion into inactive ferrous state. Depending upon the class of inhibitor, reversible or irreversible LOX inhibition can be achieved (Nelson and Seitz, 1994).
2. Iron Chelators
Substances with ability to make the complexes with protein bound ferric ion comprise a class of LOX inhibitors. Bidentate Catechols and hydroxamic acids are the main examples of inhibitors. Many of these inhibitors possess an additional reducing ability, which further strengthens their inhibitory potential. There are some substances which are pure complex-building inhibitors. The Catechols with electron withdrawing groups form stable ferric complexes where these with electron-rich substituents are rather prone to reducing the iron. (Nelson and Seitz, 1994).
3. Substrate Analogues
Substrate analogues are the compounds that mimic the fatty acids and inhibit the LOX enzyme in a time-dependant manner. The majority of these types of compounds contain acetylenic groups which are responsible for the irreversible inactivation of the enzyme. Although the mechanism of action is not fully understood, it is supposed that highly reactive allene hydro peroxides covalently bind with methionine residue in the catalytic center of enzyme, thus inhibiting the enzyme irreversibly. The prototype of this group of inhibitors which are widely used include 5,-8,-11,-14-eicosateraeonic acids. Dehydroarachidic acids (DHA) are modified derivatives of arachidonic acid in which the single acetylenic moiety is positioned according to the targeted LOX bond position in the targeted LOX subfamily, e.g., 14, 15-DHA inhibits 15-LOX selectively just as 14,15-DHA inhibits the 12-LOX (Corey, 1982). The carboxyalkyl benzyl propagyle ether derivatives combine to form another group of substrate analogues which exhibit 12-LOX inhibition (Gorins et al., 1996).
4. LOX-Activating Protein Inhibitors
A novel candidate for the inhibition of 5-LOX is an activating protein known as flap. The protein activating arachidonic acid from the transformation by LOX. (Sala et al., 1996). Two types of compounds have been found to inhibit the flap, the indol and quinoline derivatives (Mancini et al., 1992).
5. EGF-Receptor inhibitor
Experimental studies of tumor cell lines have shown that the stimulation of epidermal growth factor receptors (EGF-R) result in EGF mediated induction of 12-LOX expression and enzymatic activities. Substances those are able to inhibit EGF-R may also exhibit indirect, highly selective inhibition of 12-LOX may be used as antimetastatic agents in the future. The compounds such as 4, 5-dianilin nophthalimide were found to inhibit EGF-R in a dependant manner, leading to a significant decrease in the cellular 12-LOX protein expression (Hagmann and Borger, 1997).
Methods to Determine Lipoxygenase Activity
Various methods have been reported in the literature on the determination of lipoxygenase activity. Some are given below.
1. Spot test for lipoxygenase activity
A test for LOX is described which is adopted from a procedure for the detection of LOX bands in polyacrylamide gels. The test is an iodometric test for peroxides. It is simple and rapid method which has been used in locating lipoxygenase activity in the effluent of chromatographic columns and from a continuous preparative electrophoresis apparatus during the purification of LOX from milled wheat products (Wallace and Wheeler, 1972)
2. Ferrous oxidation-Xylenol orange assay
A. Spectrophotometric Assay
In this method the kinetics of 5(S)-hydroxyperoxy-6-trans-8, 11, 14-cis-eicosatetraenoic acid production is measured by the increase in absorbance at 234nm upon the incubation of lysates with arachidonic acid or linoleic acid (Tappel, 1953; Sik Cho et al., 2006).
B. FOX Assay
FOX assay is based on the complex formation of Fe3+ / xylenol orange and measurement of absorbance in the visible region. The absorbance spectra of mixtures of FOX reagents and 5-HEPTE are scanned under visible light ranging from 350 to 800nm (Sik Cho et al., 2006).
3. Fluorescence-based Enzyme Assay
This assay is based on the oxidation of the substrate 2,7-dichlorodihydro-fluorescein diacetate to the high fluorescent 2,7-dicholorofluorescein product. Non- specific ester cleavage of the acetate groups in 2, 7-dichlorodihydro-fluorescein diacetate is required prior to oxidation. This is achieved through the use of crude cell lysates preparation of human 5-lipoxygenase (Robert et al., 2007).