Example of enzyme competitive inhibitors. A reaction catalyzing by fumarate hydratase A and comparison of structure of fumarate substrate of reaction and maleate enzyme competitive inhibitor B [ 16 ]. Some reversible inhibitors bind so tightly to the enzyme that they are essentially irreversible. It is known that proteolytic enzymes of the gastrointestinal tract are secreted from the pancreas in an inactive form.
Their activation is achieved by restricted trypsin digestion of proenzymes. To stop activation of proteolytic enzymes, the pancreas produces trypsin inhibitor. It is a small protein molecule it consists of 58 amino acid residues [ 17 ]. This inhibitor binds directly to trypsin active site with Kd value that is equal to 0. The binding is almost irreversible; complex EI does not dissociate even in solution of 6 M urea.
The inhibitor is a very effective analog of trypsin substrates; amino acid residue Lys of inhibitor molecule interacts with aspartic residue located in a pocket of enzyme surface destined for substrate binding, thereby preventing its binding and conversion into the product Figure 4.
Structure of complex pancreatic trypsin inhibitor—trypsin and free trypsin inhibitor [ 17 ]. To obtain information concerning the mechanism of enzyme reaction, we should determine functional groups that are required for enzyme activity and located in enzyme active site. First approach is to reveal a 3D structure of enzyme with bound substrate using X-ray crystallography.
It can covalently bind to reactive groups of enzyme active site that allow to elucidate functional amino acid residues of the site. Modified amino acid residues may be found later after achievement of complete enzyme inhibition, enzyme proteolysis, and identification of labeled peptide s. Irreversible inhibitors that can be used with this aim may be divided into two groups: 1 group-specific reagents for reactive chemical groups and 2 substrate analogs with included functional groups that are able to interact with reactive amino acid residues.
These compounds can covalently modify amino acids essential for activity of enzyme active site and in such a manner can label them. One from the most known group-specific reagent that was used to label functional amino acid residue of enzyme active site of protease chymotrypsin was diisopropyl phosphofluoridate [ 18 ].
It modified only 1 from 28 serine residues of the enzyme. It means that this serine residue is very reactive. Location of Ser in active site of chymotrypsin was confirmed in investigation carried out later, and the origin of its high reactivity was revealed.
Diisopropyl phosphofluoridate was also successfully used for identification of a reactive serine residue in the active site of acetylcholinesterase [ 12 ]. To reveal reactive SH-group in active site of various enzymes, different SH-reagents were used, among them 14 C-labeled N-ethylmaleimide, iodoacetate, and iodoacetamide. Using these reagents, cysteines were revealed in the active sites of some dehydrogenase, cysteine protease, and other enzymes.
The second approach is the application of reactive substrate analogs. These compounds are structurally similar to the substrate but include chemically reactive groups, which can covalently bind to some amino acid residues. Substrate analogs are more specific than group-specific reagents. Tosyl-L-phenylalanine chloromethyl ketone, a substrate analog for chymotrypsin that is able to bind covalently with histidine residue and irreversibly inhibit enzyme, makes possible identification of Hys in chymotrypsin active site [ 19 ].
Many cellular enzyme inhibitors are proteins or peptides that specifically bind to and inhibit target enzymes. Numerous metabolic pathways are controlled by these specific compounds that are synthesized in organisms. Very interesting example of these inhibitors is protein serpins. It is a large family of proteins with similar structures. Most of them are inhibitors of chymotrypsin-like serine protease [ 20 , 21 ].
Serine proteases e. Cleavage of peptide bond by these proteases is a two-step process. This results in the release of new N-terminal part of protein substrate first product and in the formation of a covalent ester bond between the enzyme and the second part of substrate see Ref.
The second step of catalysis of usual substrates leads to the hydrolysis of ester bond and to the release of the second product C-terminal part of protein substrate. If serpin is cleaved by a serine protease, it undergoes conformational transition before the hydrolysis of ester bond between enzyme and the second part of substrate serpin. Therefore, serpins are irreversible inhibitors with unusual mechanism of action. Most immobile organisms like plants and some sea invertebrates use different poisons to defense themselves from being eaten; some vertebrates like snakes and invertebrates e.
If we will analyze the composition of these poisons, we can find in their content a lot of various enzyme inhibitors. They were selected during the evolution to stop many metabolic processes in organisms of victims that lead to their death.
Poisons of plants and invertebrates were used as medicine drugs during thousands of years. But only in the twentieth century, it became clear that the poisons contain various enzyme inhibitors as well as the blockers of some other biological molecules channels, receptors, etc.
For example, bee venom includes melittin, peptide containing 28 amino acids. This peptide can interact with many enzymes suppressing their activities; in particular, it binds with protein calmodulin [ 22 ] that are activator of many enzymes.
Special studies have shown that melittin structure imitates structure of some proteins to be exact, some part of protein molecules that can interact with target enzyme to provide their biological function [ 23 ]. Another example of natural inhibitors is cardiotonic steroids that were found initially in plants digoxin, digitonin, ouabain and in the mucus of toads marinobufagenin, bufotoxin, etc.
In the end of the twentieth century, it was shown that cardiotonic steroids are presented in low concentrations in the blood of mammals including human beings. The increase of concentration of these compounds in the blood may be involved in the development of several cardiovascular and renal diseases including volume-expanded hypertension, chronic renal failure, and congestive heart failure [ 24 ].
Natural poisons are a powerful instrument for investigation of enzyme function, and analysis of their action is necessary for these studies. It might be also a model for design of new inhibitors and activators that will imitate natural compounds with such properties. We have mentioned above nonsteroidal anti-inflammatory drugs that are the inhibitors of cyclooxygenase. This group of compounds the most prescribed drugs in the world, the oldest among them is aspirin was successfully used for more than one century around the whole world for treatment of patients with fever, cardiovascular diseases, joint pain, etc.
Among these drugs are both irreversible and reversible inhibitors that slow down production of prostaglandins that control many aspects of inflammation, smooth muscle contraction, and blood clotting. But there are many other groups of drugs that are by nature of inhibitors of some enzymes; the following groups of enzyme inhibitors are developed now by pharmaceutical companies and have very important therapeutic significances [ 24 ].
Inhibitors of angiotensin-converting enzyme ACE. ACE catalyzes a conversion of inactive decapeptide angiotensin I into angiotensin II by the removal of a dipeptide from the C-terminus of angiotensin I.
Angiotensin II is a powerful vasoconstrictor. Inhibition of ACE results in the decrease of angiotensin I concentration and in the relaxation of smooth muscles of vessels.
Inhibitors of ACE are widely used as drugs for treatment of arterial hypertension [ 25 ]. Proton pump inhibitors PPIs. Proton pump is an enzyme that is located in the plasma membrane of the parietal cells of stomach mucosa. It is a P-type ATPase that provides proton secretion from parietal cells in gastric cavity against the electrochemical gradient using energy of adenosine triphosphate ATP cleavage.
PPIs are groups of substituted benzopyridines that in acid medium of stomach are converted into active sulfonamides interacting with cysteine residues of pump [ 26 ]. Therefore, PPIs are acid-activated prodrugs that are converted into drugs inside the organisms. PPIs are introduced in therapeutic practice in 80th years of the twentieth century. Since this time, the drugs are successfully used for treatment of gastritis, gastric and duodenal ulcer, and gastroesophageal reflux disease.
Statins represent a group of compounds that are analogs of mevalonic acid. They are inhibitors of 3-hydroxymethylglutaryl-CoA reductase, an enzyme participating in cholesterol synthesis.
Statins are used as drugs preventing or slowing the development of atherosclerosis [ 27 ]. Because of the existence of some adverse effects, statins may be recommended for patients that cannot achieve a decrease of cholesterol level in the blood through diet and changes in lifestyle.
Antibiotic penicillin covalently modifies the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls and thus killing the bacteria [ 28 ].
Methotrexate is a structural analog of tetrahydrofolate, a coenzyme for the enzyme dihydrofolate reductase, which catalyzes necessarily step in the biosynthesis of purines and pyrimidines.
Methotrexate binds to this enzyme approximately fold more tightly than the substrate and inhibits nucleotide base synthesis. It is used for cancer therapy [ 29 ]. New promising direction of anticancer therapy that is connected with suppression of protein kinases controlling the cellular response to DNA damage is now on the step of development.
Selective inhibitors of these enzymes are now being tested in clinical trials in cancer patients [ 30 ]. Breakthrough in treatment of patients with acquired immune deficiency syndrome AIDS that is provoked by human immunodeficiency virus HIV was achieved recently using two different types of enzyme inhibitors.
Nucleoside reverse transcriptase inhibitors and protease inhibitors are now recommended for treatment of patients with this decease. These inhibitors affect also some other viral infections and demonstrated anticancer activity. Control of Metabolism Through Enzyme Regulation Cellular needs and conditions vary from cell to cell and change within individual cells over time. Figure: Enzyme inhibition : Competitive and noncompetitive inhibition affect the rate of reaction differently.
Competitive inhibitors affect the initial rate, but do not affect the maximal rate, whereas noncompetitive inhibitors affect the maximal rate. Competitive and Noncompetitive Inhibition The cell uses specific molecules to regulate enzymes in order to promote or inhibit certain chemical reactions.
Figure: Allosteric inhibitors and activators : Allosteric inhibitors modify the active site of the enzyme so that substrate binding is reduced or prevented. In contrast, allosteric activators modify the active site of the enzyme so that the affinity for the substrate increases. Allosteric Inhibition and Activation In noncompetitive allosteric inhibition, inhibitor molecules bind to an enzyme at the allosteric site. Cofactors and Coenzymes Many enzymes only work if bound to non-protein helper molecules called cofactors and coenzymes.
Figure: Vitamins : Vitamins are important coenzymes or precursors of coenzymes and are required for enzymes to function properly.
Multivitamin capsules usually contain mixtures of all the vitamins at different percentages. Enzyme Compartmentalization In eukaryotic cells, molecules such as enzymes are usually compartmentalized into different organelles.
Feedback Inhibition in Metabolic Pathways Feedback inhibition is when a reaction product is used to regulate its own further production. Figure: Feedback inhibition : Metabolic pathways are a series of reactions catalyzed by multiple enzymes.
Feedback inhibition, where the end product of the pathway inhibits an earlier step, is an important regulatory mechanism in cells. In noncompetitive inhibition also known as allosteric inhibition , an inhibitor binds to an allosteric site; the substrate can still bind to the enzyme, but the enzyme is no longer in optimal position to catalyze the reaction. Feedback inhibition involves the use of a reaction product to regulate its own further production. Inorganic cofactors and organic coenzymes promote optimal enzyme orientation and function.
Vitamins act as coenzymes or precursors to coenzymes and are necessary for enzymes to function. Metabolic reactions, such as anabolic and catabolic processes, must proceed according to the demands of the cell.
In order to maintain chemical equilibrium and meet the needs of the cell, some metabolic products inhibit the enzymes in the chemical pathway while some reactants activate them. Feedback inhibition : Metabolic pathways are a series of reactions catalyzed by multiple enzymes. Feedback inhibition, where the end product of the pathway inhibits an earlier step, is an important regulatory mechanism in cells.
The production of both amino acids and nucleotides is controlled through feedback inhibition. For an example of feedback inhibition, consider ATP. It is the product of the catabolic metabolism of sugar cellular respiration , but it also acts as an allosteric regulator for the same enzymes that produced it.
This feedback inhibition prevents the production of additional ATP if it is already abundant. Enzymes catalyze chemical reactions by lowering activation energy barriers and converting substrate molecules to products.
Enzymes bind with chemical reactants called substrates. There may be one or more substrates for each type of enzyme, depending on the particular chemical reaction. In some reactions, a single-reactant substrate is broken down into multiple products.
In others, two substrates may come together to create one larger molecule. Two reactants might also enter a reaction, both become modified, and leave the reaction as two products. Since enzymes are proteins, this site is composed of a unique combination of amino acid residues side chains or R groups.
Each amino acid residue can be large or small; weakly acidic or basic; hydrophilic or hydrophobic; and positively-charged, negatively-charged, or neutral. The positions, sequences, structures, and properties of these residues create a very specific chemical environment within the active site.
A specific chemical substrate matches this site like a jigsaw puzzle piece and makes the enzyme specific to its substrate. Increasing the environmental temperature generally increases reaction rates because the molecules are moving more quickly and are more likely to come into contact with each other.
However, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the enzyme and change its shape. If the enzyme changes shape, the active site may no longer bind to the appropriate substrate and the rate of reaction will decrease.
Dramatic changes to the temperature and pH will eventually cause enzymes to denature. This model asserted that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view called induced fit.
Induced Fit : According to the induced fit model, both enzyme and substrate undergo dynamic conformational changes upon binding. The enzyme contorts the substrate into its transition state, thereby increasing the rate of the reaction. When an enzyme binds its substrate, it forms an enzyme-substrate complex. This complex lowers the activation energy of the reaction and promotes its rapid progression by providing certain ions or chemical groups that actually form covalent bonds with molecules as a necessary step of the reaction process.
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