**3. Enzyme activators**

#### **3.1. Definition and mechanisms of action**

Enzyme *activators* are chemical compounds that increase a velocity of enzymatic reaction. Their actions are opposite to the effect of enzyme inhibitors. Among activators we can find ions, small organic molecules, as well as peptides, proteins, and lipids.

There are many enzymes that are specifically and directly activated by small inorganic molecules, mainly by cations such as Ca2+ which is a the second messenger (among enzymes activated by Ca2+, we can find different regulatory enzymes, in particular phospholipases II, protein kinases C, adenylyl cyclases, etc.). These enzymes usually have special site for Ca2+ binding; the binding of Ca2+ with it results in the change of enzyme conformation that increase enzyme activity [33].

Cations can bind not only with enzyme but also with the substrate increasing its affinity to the enzyme that activate enzyme. For example, magnesium ions interact with ATP or with other nucleotides that are negatively charged molecules, decreasing their charge that provides effective binding of nucleotides in substrate binding site of various enzymes and increasing their activity.

In some cases, activation of enzymes is due to the elimination of enzyme inhibitors. In total this effect looks as enzyme activation. Some cations including heavy metal cations inhibit definite enzymes. Small organic compounds like ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′ tetraacetic acid (EGTA) and ethylenediaminetetraacetic acid (EDTA) that are known as chelating agents bind these inhibitory cations and by this way can eliminate their inhibitory effect.

Special group of activators can produce activation of target enzymes only after the formation of complex with another molecule. This complex, in turn, binds to enzyme and increases the velocity of enzymatic reaction. The most well-known example of such type of activators is Ca-binding protein calmodulin (calcium-modulated protein) that is expressed in all eukaryotic cells. Calmodulin is a small protein containing 148 amino acids (16.7 kDa). Its molecule consists of two symmetrical globular domains each with two Ca-binding motifs (EF-hand) located on N- and C-domains that are jointed by flexible linker. Flexibility of calmodulin molecule and the presence of nonpolar grooves in the middle part of the protein allow it to bind a large variety of proteins [33]. The binding of Ca2+ to calmodulin changes its conformation. These, in turn, make complex calmodulin-Ca2+ suitable for interaction with target enzymes (calmodulin-dependent protein kinases and phosphatases,Ca-ATPase of plasma membrane, etc.), by this manner increasing their activity. Therefore calmodulin is considered as a participant of calcium signal transduction pathway that provides enforcing and prolongation of the effect of Ca2+ as a second messenger [34].

#### **3.2. Allosteric enzyme modulators**

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,

Statins represent a group of compounds that are analogs of mevalonic acid. They are inhibitors of 3-hydroxy-3-methylglutaryl-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

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 1000-fold more tightly than the

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. Presented here list of enzyme inhibitors that are used in therapy of numerous deceases that is far from being complete. But even mentioned above, points demonstrate how useful and important are therapeutic application of theoretical knowledge obtained as result of study of enzyme inhibitors [31]. Sciences around the world are involved in a search of new inhibitors of known enzymes that have therapeutic significance. An example of this complex research is a work devoted to design, synthesis, and study of new inhibitors of carbonic anhydrase, an enzyme that is involved in the development of such symptoms and deceases as edema, glaucoma, obesity,

Enzyme *activators* are chemical compounds that increase a velocity of enzymatic reaction. Their actions are opposite to the effect of enzyme inhibitors. Among activators we can find

ions, small organic molecules, as well as peptides, proteins, and lipids.

substrate and inhibits nucleotide base synthesis. It is used for cancer therapy [29].

gastric and duodenal ulcer, and gastroesophageal reflux disease.

252 Enzyme Inhibitors and Activators

synthesis of bacterial cell walls and thus killing the bacteria [28].

cancer, epilepsy, and osteoporosis (see Ref. [32]).

**3.1. Definition and mechanisms of action**

**3. Enzyme activators**

Inhibitors and activators (modulators) that bind to enzymes not in the active site but in special center located far enough from it have name allosteric modulators. Their binding to allosteric sites induces the change of enzyme conformation that affects both the structure of active site and enzyme conformational mobility leading to the decrease or to the increase of enzyme activity. Just as enzyme active site is specific in relation to substrate, the allosteric site is specific to its modulator [16].

Many metabolic pathways are regulated through the action of allosteric modulators. Enzymes in metabolic pathways work sequentially, and in such pathways, a product of one reaction becomes a substrate for the next one. The rate of whole pathway is limited by the rate of the lowest reaction. Allosteric regulators often are a final product of whole metabolic pathway that activates enzymes catalyzing a limiting step of the whole pathway. Enzymes in a metabolic pathway can be inhibited or activated by downstream products. This regulation represents negative and positive feedbacks that slow metabolic pathway when the final product is produced in large amounts or accelerate it when a final product is presented in low concentration. Therefore, allosteric modulators are important participants of such negative and positive feedbacks in metabolic pathways or between them making metabolism self-controlled.

For example, ATP and citrate are inhibitors of phosphofructokinase that is a key enzyme of glycolytic pathway. One product of glycolysis is ATP. Another product is pyruvate that after the conversion into acetyl-CoA is condensed with citrate opening cycle of citrate acids (Krebs cycle). Reactions of this cycle produce reduced nicotinamide adenine dinucleotide reduced (NADH) and flavinadeninidinucleotide reduced (FADH2), oxidation of which is coupled with massive production of ATP in mitochondria. Availability of ATP or citrate inhibits glycolysis preventing glucose oxidation (negative feedback). Inhibition of phosphofructokinase by ATP or by citrate occurs by allosteric manner [35]. Described negative feedback control maintains a steady concentration of ATP in the cell. It should be noted also that metabolic pathways are regulated not only through inhibition but also through activation of the key enzymes. Mentioned above phosphofructokinase is activated by adenosine diphosphate (ADP), adenosine monophosphate (AMP), and fructose-2,6-bisphospate that represents positive feedback control.

Enzymes that are regulated by allosteric modulators are usually presented by several interacting subunits (they are called oligomers). A very interesting example of regulation of the activity of oligomeric enzymes is c-AMP-dependent protein kinase that is an important regulatory enzyme participating in the phosphorylation of serine and threonine residues of target proteins changing by this way their activity. This enzyme consists of four subunits; two of them are catalytic and two are regulatory. Cyclic AMP (c-AMP) is allosteric activator of this enzyme. Catalytic subunit being bound to the regulatory one is inactive. Binding of two c-AMP molecules to allosteric sites of each regulatory subunit induces their conformation transition that results in dissociation of the tetrameric complex and in activation of catalytic subunits [36]. Decrease of c-AMP concentration leads to its dissociation from the allosteric site and to association of regulatory and catalytic subunits with subsequent inactivation of catalytic subunits. By this way, c-AMP activity depends upon the c-AMP concentration in the cell.
