Enzyme activators are molecules that bind to enzymes and increase their activity. They are the opposite of enzyme inhibitors. These molecules are often involved in the allosteric regulation of enzymes in the control of metabolism. An example of an enzyme activator working in this way is fructose 2,6-bisphosphate , which activates phosphofructokinase 1 and increases the rate of glycolysis in response to the hormone insulin.
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Enzymes are very effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Enzyme inhibitors and activators that modulate the velocity of enzymatic reactions play an important role in the regulation of metabolism. Enzyme inhibitors are also useful tool for study of enzymatic reaction as well as for design of new medicine drugs. In this chapter, we focused on the properties of enzyme inhibitors and activators. Here we present canonical inhibitor classification based on their kinetic behavior and mechanism of action.
We also considered enzyme inhibitors that were used for design of various types of pharmacological drugs and natural inhibitors as a plausible source for design of future drugs. Mechanisms of action of enzyme activators and some features of allosteric modulators are considered. Enzyme Inhibitors and Activators.
Enzymes E is a group of biologically active polymers mainly proteins that catalyze almost all metabolic reactions in all living organisms. Enzymes are able to accelerate chemical reaction dividing it into separate steps. According to contemporary hypothesis, high conformational mobility of the enzymes allows them to adopt their active sites to substrate s and intermediates of the reaction in the best way [ 1 , 2 ].
Multiple conformers of enzymes with close values of free energy preexist in the solution simultaneously. Along the reaction way, a conformer is picked out, the structure of which can stabilize definite intermediate that makes a reaction more thermodynamically profitable [ 3 ]. By this way, inhibitors stop enzymatic reaction. On the other hand, the binding of enzyme activators may lead to the creation of more profitable conformers that can be more effective in carrying out definite steps of the reaction.
Therefore, they will accelerate enzymatic reaction. Taking into account this information about enzymes in this chapter, we consider contemporary knowledge about enzyme inhibitors and activators.
Enzymes are different chemical compounds that are combined into a group because of their only feature—they can suppress enzyme activity. The suppression of the activity is the result of the binding of inhibitor to the enzyme molecule that arrests catalytic reaction. Because enzymes catalyze most part of chemical reactions in living organisms, the enzyme inhibitors play an important role in the development of different sciences biochemistry, physiology, pharmacy, agriculture, ecology as well as the technologies production of pharmaceutical drugs, insecticides, pesticides, chemical weapons, etc.
Many pharmacological drugs are enzyme inhibitors. The group of well-known pharmaceutical agents with name nonsteroidal antiinflammatory drugs NSAIDs includes inhibitors of enzyme cyclooxygenase that catalyzes a first step of synthesis of biologically active compounds prostaglandins that are responsible for the development of pain, inflammation, fever, contraction of smooth muscle, formation of blood clots, and others [ 5 ].
However, this classification does not reflect mechanism of their interaction with enzyme. In accordance with the mode of action, enzyme inhibitors may be divided into two different groups reversible and irreversible inhibitors. Reversible inhibitors, in turn, may be combined in four groups in accordance with kinetic behavior competitive, uncompetitive, noncompetitive, and mixed inhibitors [ 6 ].
The mechanism of action of enzyme inhibitors includes a step of enzyme-inhibitor complex formation EI complex that has no or low enzyme activity. An irreversible inhibitor dissociates from this complex very slow because it is tightly bound to the enzyme. Mainly this mode of inhibition is connected with the formation of covalent bond or hydrophobic interaction between enzyme and inhibitor.
Irreversible inhibitors usually react with the enzyme and change it chemically. These inhibitors often contain reactive functional groups that modify amino acid residues of enzyme that are essential for its activity. They also can provide inhibition affecting the enzyme conformation. An example of irreversible inhibitor is N-ethylmaleimide that covalently interacts with SH-group of cysteine residues of enzyme molecules, like peptidase insulin-degrading enzyme [ 7 ], 3-phosphoglyceraldehyde dehydrogenase [ 8 ], or hydrophobic compound from group of cardiotonic steroids that at the last bind to Na,K-ATPase using hydrophobic interactions [ 9 ].
Another well-known irreversible inhibitor is diisopropyl phosphofluoridate that modifies OH-group of serine residue in active site of such enzymes as chymotrypsin and other serine proteases [ 10 , 11 ] or acetylcholine esterase in cholinergic synapsis of the nervous system being a potent neurotoxin [ 12 ]. Inhibition of this enzyme causes an increase in the acetylcholine neurotransmitter concentration that results in muscular paralysis and death. Inhibitor of cyclooxygenase aspirin acetyl salicylic acid covalently modifies OH-group of serine residue located in a close proximity to the active site of cyclooxygenase [ 13 ].
Irreversible inhibition is different from irreversible enzyme inactivation. Irreversible inhibitors are generally specific for one class of enzymes and do not inactivate all proteins. In contrast to denature agents such as urea, detergents do not destroy protein structure but specifically alter the active site of the target enzyme. Consequently because of tight binding, it is difficult to remove an irreversible inhibitor from the EI complex after its formation [ 14 ].
So, we can refer some chemical compound to irreversible enzyme inhibitor, if after the formation of EI complex, the dilution of it with significant amount of water — excess does not restore enzyme activity.
Irreversible inhibitors display time-dependent loss of enzyme activity. Interaction of irreversible inhibitor with enzyme is a bimolecular reaction:. However, usually the action of irreversible inhibitors is characterized by the constant of observed pseudo-first order reaction under conditions when concentration of inhibitor is significantly higher than concentration of the enzyme.
Tangent of slope angle of straight line obtained by this way will be equal to value of constant of pseudo-first order inhibition. The value of rate constant of bimolecular reaction for irreversible inhibition may be then calculated by dividing the obtained value of constant of pseudo-first order reaction per inhibitor concentration.
Reversible inhibitor binds to the enzyme reversibly [ 6 , 14 ]. It means that there is equilibrium between the formation and dissociation of EI complex:. Usually reversible inhibitor binds to the enzymes using non-covalent interactions such as hydrogen or ionic bonds. Different types of reversible inhibition are produced depending on whether these inhibitors bind to the enzyme, the enzyme-substrate complex, or both.
One type of reversible inhibition is called competitive inhibition. In this case, there are two types of complexes: enzyme inhibitor EI and enzyme substrate ES ; complex EI has no enzyme activity. The substrate and inhibitor cannot bind to the enzyme at the same time. This inhibition may be reversed by the increase of substrate concentration. However, the value of maximal velocity Vmax remains constant.
It can be competitive inhibition not only in relation to substrate but also to cofactors, as well as to activators. Kinetic test for reversible inhibitor classification. Another type of reversible inhibition is uncompetitive inhibition. In this case, the inhibitor binds only to the substrate-enzyme complex; it does not interfere with the binding of substrate with active site but prevents the dissociation of complex enzyme substrate: it resulted in the dependence of the inhibition only upon inhibitor concentration and its Ki value.
The third type of inhibition is noncompetitive. This type of inhibition results in the inability of complex enzyme E inhbitor I substrate EIS to dissociate giving a product of reaction. In this case, inhibitor binds to E or to ES complex.
The binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. As a result, the extent of the inhibition depends only upon the concentration of the inhibitor. In some cases, we can see mixed inhibition, when the inhibitor can bind to the enzyme at the same time as to enzyme-substrate complex.
However, the binding of the inhibitor effects on the binding of the substrate and vice versa. This type of inhibition can be reduced, but not overcome by the increase of substrate concentrations. Although it is possible for mixed-type inhibitors to bind in the active site, this inhibition generally results from an allosteric effect of inhibitor see below.
Special case of enzyme inhibition is inhibition by the excess of substrate or by the product. This inhibition may follow the competitive, uncompetitive, or mixed patterns. Inhibition of enzyme by its substrate occurs when a dead-end enzyme-substrate complex forms. Often in the case of substrate inhibition, a molecule of substrate binds to active site in two points e. An example of such inhibition is inhibition of acetyl cholinesterase by the excess of acetylcholine [ 15 ].
Enzyme inhibition by substrate. Productive binding of one substrate molecule with two points of enzyme active site A and unproductive binding of two substrate molecules with the same site B. Competitive inhibitors mainly interact with enzyme active site preventing binding of real substrate.
Enzyme is highly stereospecific; it catalyzes the hydration of the trans-double bound of fumarate but not maleate cis-isomer of fumarate. Maleate binds to active site with high affinity preventing the binding of fumarate. Despite the binding maleate to active site, it cannot be converted into the product of reaction.
However, maleate occupies active site making it inaccessible for real substrate and providing by this way the inhibition [ 16 ]. 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 .
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.
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.
Enzyme Inhibitors and Activators
EFFECT OF ENZYME INHIBITORS AND ACTIVATORS ON THE MULTIPLICATION OF TYPHUS RICKETTSIAE
By injection into typhus-infected yolk sacs, a number of agents were tested for possible inhibition or acceleration of rickettsial growth. The previously reported rickettsiostatic activity of penicillin was further confirmed. Para-aminobenzoic acid, in single injections of 6. No conclusion could be drawn regarding the possibility of a synergistic action of para-aminobenzoic acid and penicillin. Para-aminobenzoic acid neutralized with sodium hydroxide was found to be as effective as the acid itself, when given in single injections of 6. Sodium benzoate, as Well as the ortho and meta forms of aminobenzoic acid were found to be ineffective when given in similar amounts. Para-aminobenzoic acid, when added to the food in a concentration of 3 per cent, was shown to have a remarkably effective chemotherapeutic action on murine typhus infection in mice.