Enzyme Inhibition Lecture Notes PDF

№11 Reversible and irreversible inhibition The present lecture material was mainly prepared by Prof. Ana Maneva, DSc, Assoc. Prof. Anelia Bivolarska, Ph.D., MD, and Prof. Tatyana Vlaykova, Ph.D., from the Department of Medicinal Biochemistry at the Medical University - Plovdiv. Non-specific inhibi...

№11 Reversible and irreversible inhibition The present lecture material was mainly prepared by Prof. Ana Maneva, DSc, Assoc. Prof. Anelia Bivolarska, Ph.D., MD, and Prof. Tatyana Vlaykova, Ph.D., from the Department of Medicinal Biochemistry at the Medical University - Plovdiv. Non-specific inhibition – in case of drastic effects causing denaturation (acids, bases, temperature, heavy metals, etc.). Specific - reversible and irreversible. Reversible is of three main types: competitive, non-competitive and uncompetitive.  Irreversible inhibition ✓ If the inhibitor irreversibly binds to the enzyme – e.g. through a covalent bond, the kinetic characteristic will be similar to noncompetitive inhibition because the end effect is a loss of enzyme activity. ✓ Unlike reversible non-competitive inhibition, in irreversible inhibition dissociation of the enzyme-inhibitor complex and restoration of enzyme activity cannot be achieved.  Examples of irreversible inhibitors 1. Military Poisons 2. An example of irreversible inhibition under physiological conditions: Zymogens (proenzymes) are precursors of the active enzyme, where the active site is blocked with part of the polypeptide chain itself. In order to obtain the active enzyme, it is necessary to react with another proteolytic enzyme to remove the blocking fragment (limited proteolysis). 3. Another example is the protein avidin, which is an egg white that forms a non-dissociable complex with biotin, the prosthetic group of carboxylases.  Mechanism of action of irreversible inhibitors Group of active site Irreversible inhibitors Enzyme affected reacting with the inhibitor iodoacetamide SH group of the Thiol-dependent p-chlormercury benzoate enzymes enzyme arsenic derivatives agents blocking aldehyde groups: enzymes, acting with coenzyme -CHO group of the cyanides, sodium bisulfide, etc. pyridoxal phosphate coenzyme diazopropylfluorine phosphate serine proteases, esterases, -OH group of the enzyme (military poison) e.g. acetylcholinesterase egg white against avidin carboxylases groups of the prosthetic biotin group  4. Example: Aspirin exerts an anti-inflammatory effect as an irreversible inhibitor COX-2 leads to the formation of inflammatory prostaglandins. Aspirin irreversibly inhibits the enzyme cyclooxygenase 2 (COX-2) and suppresses the transcription of the gene encoding COX-2, thereby preventing the action of this enzyme associated with the inflammatory response by two mechanisms. Basically, it suppresses more COX-1. Aspirin forms a covalent bond with a serine residue of the COX-2 enzyme, to which an acetyl residue from aspirin attaches (acetylates).  5. Example: Suicide substrates — a type of irreversible inhibition Suicide substrates, also called Trojan horse substrates, are of the affinity tag type. As substrate analogs, they bind specifically and with high affinity covalently to the active site of the enzyme. Penicillin is a suicide substrate. The antibiotic penicillin, exerting its effect by covalently binding to a serine residue in the active site of the enzyme glycoprotein peptidase, forms cross-links in peptidoglycan chains during the synthesis of the bacterial wall. Blocking cell wall synthesis renders bacterial cells susceptible to rupture by osmotic lysis and bacterial growth is inhibited. Irreversible inhibition Penicillin consists of a thiazolidine ring joined by a β- lactam ring to a variable group R. A reactive peptide bond in the β-lactam ring is covalently attached to a serine residue in the active site of the glycoprotein peptidase. The penicinoyl-enzyme complex is catalytically inactive. The bond between the enzyme and penicillin is stable, penicillin is irreversibly bound.  Suicide инхибитори Suicide инхибитори приличат на субстрата, но атакуват ензима, когато е активиран. 5-флуороурацилът (който се превръща в тялото в 5F-дУМФ) е един suicide инхибитор на тимидилат синтазата, и блокира DNA синтезата в раковите клетки 5-флуорурацил Reversible competitive and non- competitive inhibitors Competitive inhibition  with inhibitor Slope= without inhibitor 1. Km is increased 2. Vmax is not changed The inhibitor is a structural analogue of the substrate. Therefore: ✓ The inhibitor reacts only with free enzyme. ✓ ES and EI complexes are obtained. ✓ Inhibition is reversible and can be eliminated by adding high concentrations of substrate.  Graphical representation of the Lineweaver and Burk equation in the presence of a competitive inhibitor In competitive inhibition, the Km is increased (an expression of reduced affinity between the substrate and the enzyme, since the substrate can be replaced by an analog). Because inhibitor and substrate compete for the same binding site, high concentrations of substrate can completely displace the inhibitor from the active site and restore Vmax. Therefore, with this type of inhibition, Vmax does not differ from that of the reaction without inhibitor.  Malonate, as a competitive inhibitor of succinate succinate succinate dehydrogenase dehydrogenase E-FADH2 FAD fumarate succinate malonate Through the two carboxyl groups, malonate binds to the active site of the enzyme, but since it does not have two adjacent methylene groups, it cannot be dehydrogenated by the catalytic action of succinate dehydrogenase.  Antimetabolites - competitive inhibitors Medicines (antiviral, antibacterial, antitumor) are purposefully synthesized with a view to inhibiting a certain enzyme in a given metabolic pathway, with the aim of having limited toxicity for the patient. Toxicity is difficult to avoid because, with the exception of cell wall biosynthesis in bacteria, there are few metabolic pathways unique to tumors, viruses, or bacteria. Therefore, drugs that kill microorganisms are also harmful to human cells, and this must be taken into account when dosing and timing! But in all these cases, the higher sensitivity of microorganisms or tumor cells to antimetabolites is relied upon, compared to that of the macroorganism.  Structure of some antimetabolites: sulfanilamide - antimetabolite of p-aminobenzoic acid; allopurinol - antimetabolite of hypoxanthine; 5-fluorouracil - antimetabolite of pyrimidine bases; 6-mercaptopurine - antimetabolite of purine bases. sulfanilamide p-aminobenzoic acid 5-fluorouracil 6-mercaptopurine hypoxanthine allopurinol The higher sensitivity of microorganisms or tumor cells may be due to the fact that: 1) antagonists affect such units of the metabolism that are key/vital for the microorganism, but are secondary or absent in the macroorganism; 2) the macroorganism has a variety of protective means that suppress the activity of the antagonist or reduce its concentration. 3) Some of these antimetabolites are substrate analogs, others are coenzyme analogs.  Puromycin Puromycin is a structural analogue of Tyr-tRNA, an inhibitor of protein biosynthesis. Puromycin structure and mechanism of action. (A) Chemical structures of the 3′ end of an aminoacylated tyrosyl-tRNA (left) and puromycin (right). The different bonds between the nucleoside and amino acid moieties are shown in pink. (B) Basic mechanism of puromycin action. During translation elongation, aa-tRNA enters the A-site and accepts the nascent polypeptide chain from the peptidyl-tRNA in the P-site. Following translocation, the A-site becomes available to accommodate the next aa- tRNA (top). Like aa-tRNA, puromycin can enter the A-site and accept the nascent chain. This results in translation termination, ribosome disassembly and release of the nascent chain bearing a 3′ puromycin (bottom). Source: https://doi.org/10.1016/j.csbj.2020.04.014  Examples of antimetabolites used in AIDS therapy Acyclovir (acycloguanosine) 3'-azido-3'-deoxythymidine Acyclovir or acycloguanosine (purine analog) and 3'-azido-3'- deoxythymidine (AZT) (pyrimidine analog).  Sulfonamides Sulfonamides are a large class of compounds with the general formula R - SO2 - NHR’. Sulfonamide is the simplest representative. It is an antibacterial agent because it is a structural analogue of p-aminobenzoic acid. p-Aminobenzoic acid is part of a more complex compound folic acid (FA), which is necessary for the synthesis of nucleotides and nucleic acids, and therefore necessary for bacterial growth. Bacteria cannot take up FA from the host, they must synthesize it. Incorporating the sulfonamide into the FA renders it inactive and the bacteria cannot grow and divide. Sulfanilamide p-aminobenzoic acid  FA is not synthesized in humans, for them it is a (-) vitamin that is taken in Sulfonamides with food. Therefore, sulfonamides are not harmful to him in the doses that kill the bacteria. (-) methotrexate Source: DOI:10.5937/savteh1701058T Methotrexate (ametopterin) is used to treat childhood leukemia (a malignant division of white blood cells) because it is a close structural analogue of folic acid. Inhibits the reduction of folate to dihydrofolate (DHF) and tetrahydrofolate (THF). Folic acid methotrexate Allopurinol is used in the treatment of hyperuricemia and gout At first, allopurinol acts as a substrate of xanthine oxidase, and then as its inhibitor. The enzyme converts it to alloxanthin, which remains tightly bound to the active site. Therefore, such inhibitors are called suicide substrates. As a very close structural analogue of the hypoxanthine base, allopurinol acts as its "suicide substrate".  Overview on the allopurinol effect In addition to being an inhibitor of xanthine oxidase, allopurinol reduces the overall rate of purine formation in the de novo pathway because it binds part of PRPP, which is an intermediate metabolite and activator of this pathway. Reduced formation of purine nucleotides reduces the formation of uric acid. After the introduction of allopurinol, the formation of uric acid is stopped. The concentration of hypoxanthine and xanthine (more soluble) in the serum increases, and that of uric acid decreases. Purine metabolism. The metabolic scheme shows the first and rate-limiting step of de novo purine synthesis mediated by the enzyme 5'- phosphoribosyl-1- (-) pyrophosphate (PRPP) allopurinol amidotransferase, and the salvage pathway mediated by hypoxanthine phosphorybosyltransferase (HPRT) and adenine phosphorybosyltransferase (APRT). Other abbreviations: adenosine mono/diphosphate (AMP/ADP), inosine (-) monophosphate (IMP), guanosine monophosphate (-) allopurinol (GMP). HPRT catalyzes the DOI:10.1186/1750-1172-2-48 salvage synthesis of GMP.  Ethanol finds therapeutic use as a competitive inhibitor in the treatment of ethylene glycol poisoning ✓ About ½ of those who ingested ethylene glycol, which is contained in antifreeze, died. Ethylene glycol itself is not toxic. The product of its oxidation - oxalic acid - is toxic, as its crystals can cause acute kidney damage when deposited. ✓ The first step in the conversion of ethylene glycol to oxalic acid is its oxidation to an aldehyde by alcohol dehydrogenase. This reaction can be effectively inhibited by administration of non-toxic doses of ethyl alcohol. ✓The basis of this effect is the fact that ethyl alcohol is also a substrate of alcohol dehydrogenase and thus blocks the oxidation of ethylene glycol, which is excreted without further complications. ✓Ethyl alcohol can also be used as a competitive substrate for the treatment of methanol poisoning. Other examples of drugs acting as competitive inhibitors Noncompetitive inhibition  Graphical representation of the Lineweaver and Burk equation in the presence of a noncompetitive inhibitor With inhibitor Slope= Without inhibitor 1. Vmax is decreased 2. Km is not changed The inhibitor is not a structural analogue of the substrate. Therefore: ✓ The inhibitor reacts with both free enzyme and ES. ✓ ES, EI, and ESI complexes are obtained. ✓ Inhibition cannot be overcome by adding substrate. Non-competitive inhibitors bind reversibly outside the active site of the enzyme: they change the enzyme conformation allosterically and reduce the rate of formation of the final product. ✓They have no effect on Km because the substrate can bind to the active site. In this case, ESI complexes can be formed. When the inhibitor binds to the allosteric center, however, the conformation of the enzyme molecule changes and the enzyme can only bind a substrate, but not a product. ✓In this type of inhibition, Vmax decreases because part of the protein ceases to be catalytically active. These inhibitors are not substrate analogues. Since the inhibitor can bind independently of the substrate, three types of complexes can be formed, of which ESI and EI do not lead to product formation. Comparative characterization of competitive and noncompetitive inhibition  Uncompetitive reversible inhibition In noncompetitive reversible inhibition, the inhibitor binds only to the ES complex in regions other than the active site of the enzyme. With this type of inhibition, Km and Vmax decrease. As the concentration of the substrate increases, the binding of the inhibitor to the ES increases. With uncompetitive inhibitor Example: NaN3 inhibits the enzyme cytochrome oxidase. 1/Vmax´ (no I) No inhibitor -1/Km´ -1/Km 1/Vmax (with I) (no I) (no I) Enzyme activation  Activators Activators are substances that increase the rate of a catalyzed reaction. They can be enzymes, metabolites, metal ions. With metal ions, it is difficult to distinguish exactly whether the ion acts as an activator or a cofactor. This includes the cases of: conversion of inactive enzyme precursors (proenzymes) into active enzymes and allosteric activation.  1. Proenzymes ✓ Proenzymes are precursors of proteases necessary for important types of processes: digestion, blood clotting and dissolution of clots, complement system (primary defense mechanism in innate immunity). ✓ Their secretion as inactive precursors protects against their digestive proteolytic action and thus provides protection for the tissue that produces them. ✓ When they are converted into active enzymes, one or more peptides from the proenzyme chain are irreversibly proteolytically removed (limited proteolysis), which reduces the molecular weight and unblocks important groups necessary for the formation of the active center. ✓ Conformational changes and formation of the active center occur. Usually, the cleavage of the blocking peptide or peptides occurs by the action of another enzyme or autocatalytically.  Activation of the enzyme precursor trypsinogen into the active enzyme trypsin and chymotrypsinogen into chymotrypsin Trypsinogen contains a hexapeptide at the amino end, which is released under the action of enteropeptidase (peptidase from the intestinal juice).  Activation of prothrombin to thrombin https://doi.org/10.1074/jbc.M208423200 Schematic representation of human prothrombin activation. Prothrombin consists of a membrane- binding N-terminal domain (Fragment 1.2) and a C-terminal catalytic domain (Prethrombin 2). Two peptide bonds (Arg273–Thr274 (site (2)) andArg322–Ile323 (site (1))) must be hydrolyzed by factor Xa to activate prothrombin. This creates two possible pathways of activation,A (cut (2)) and B (cut (1)) as well as C (cut (1)) and D (cut (2)), and also creates two possible released intermediates, Pre2 and MzIIa. A third possible pathway of activation is envisioned (E, fast consecutive cuts (1) and (2)) in which no intermediate is released, i.e.extensive channeling occurs.  Activation of complement system proteins ✓ Both C1q and C1s are proenzymes that possess serine esterase catalytic domains, and the sequential auto-activation of C1r and C1s can lead to the activation of C4 and C2 through cleavage by C1s. ✓ Cleavage of the α chain of C4 releases C4a and C4b. ✓ C1s cleaves C2 to produce C2a and C2b. ✓ C4b2a (C3 convertase) cleaves C3, generating C3a and C3b. ✓ C4b2a3b cleaves C5 into C5a and C5b, etc. DOI:10.1007/s12026-011-8239-5 The complement system is activated in three ways: via the classical pathway, which includes the proteins C1, C4, C2, and C3; the alternative pathway, with the participation of C3 and protein factors B, D, and P; and the lectin pathway, with the participation of mannose- binding lectin (MBL and MBL-associated proteases). All three pathways lead to the activation of the C5, C6, C7, C8, and C9 proteins, resulting in the sequential assembly of C5b-7, C5b-8, and C5b-9 (membrane attack) complexes on the target cell to make pores in its cell membrane to kill it. 2. Allosteric regulation (allosteric activation and allosteric inhibition - suppression)  The word allosteric comes from the Greek allos-other and stereos- shape. 1. In addition to an active center, some enzymes also have one or more allosteric centers. Allosteric effectors are associated with them - allosteric activator or allosteric inhibitor. 2. The effector has no structural similarity to the substrate of the affected enzyme. 3. The allosteric effector causes a conformational change of the active center - the activity of the enzyme is suppressed or activated. 4. In some cases, the regulatory and active parts are of different subunits. 5. Allosteric enzymes have a quaternary structure and are regulatory enzymes. 6. Allosteric regulation is advocated in important synthesis chains - e.g. the synthesis of purine and pyrimidine nucleotides.  Cooperative effect in allosteric regulation ✓An allosteric enzyme is an oligomer whose biological activity is expressed in a change in the conformation of its quaternary structure, and the Michaelis-Menten equation is not valid for them. ✓Instead of a rectangular hyperbola, the dependence of V on [S] is a sigmoidal curve.  Homotropic and heterotropic allosteric regulation A. Homotropic regulation In many cases, the substrate induces remote allosteric effects when it binds to the catalytic site. When the substrate binds to the active site, it exerts an active modulation effect on the other subunits of the molecule. Substrates acting as effectors are called homotropic effectors. A commonly used example of a homotropic effector is hemoglobin, although it is not an enzyme and does not fit the general definition. When the substrate is also an effector, it can bind to both the active and allosteric sites. B. Heterotropic regulation The effector differs from the substrate and binds at a structurally different site.  Allosteric control when the regulatory part and the active part are on different subunits An example of such an effect is cyclic (c) AMP-dependent protein kinase A (PKA). The enzyme is a tetramer, contains 2 catalytic and 2 regulatory subunits, and is not enzymatically active. When the intracellular level of cAMP rises, one molecule of cAMP binds to each regulatory subunit and the tetramer dissociates into a regulatory dimer and 2 catalytic monomers. In the dissociated form, the catalytic subunits are fully active. They catalyze the phosphorylation of a number of other enzymes, such as those involved in the regulation of glycogen metabolism. The regulatory subunits have no catalytic activity.  Retroinhibition as a type of allosteric inhibition ✓ From a biological point of view, the most important non-competitive inhibition is allosteric inhibition, which affects processes throughout the metabolic chain. ✓ In it, the end product in the metabolic chain affects the first chain-specific enzyme, or the rate-determining enzyme, or both, if they do not match. ✓ When an initial enzyme is inhibited by the end products in a chain, the term retroinhibition or negative feedback inhibition is used. Examples: 1. Allosteric effects in the synthesis of purine nucleotides A negative sign represents the allosteric inhibition of the two regulatory enzymes by the final products - the purine nucleotides IMP (inosine monophosphate), AMP and GMP. Allosteric activation of the second enzyme amidotransferase (main regulatory enzyme of purine nucleoside synthesis; PRPP synthetase is also a regulatory enzyme) by PRPP (phosphoribosyl pyrophosphate) is represented by a positive sign.  2. Treatment of orotaturia by pyrimidine nucleotides, which as end products allosterically suppress endogenous synthesis Treatment of orotaturia by pyrimidine nucleotides, which as end products allosterically suppress endogenous synthesis. In case of deficiency of two enzymes in the biosynthesis chain of pyrimidine nucleotides: orotate phosphoribosyl transferase (E5) and orotidine-5'-phosphate decarboxylase (E6), the intermediate metabolite orotate accumulates in the blood, and from there it passes into the urine (orotaturia). The end products pyrimidine nucleotides - UTP, CTP, TDP (thymidine diphosphate - with deoxyribose) are missing, and they are necessary for the synthesis of nucleic acids. The enzyme block is bypassed by injecting UMP. De novo biosynthetic pathway of pyrimidine nucleotides in plants. Enzymes shown are: (1) Carbamoyl phosphate synthetase, (2) aspartate transcarbamoylase, (3) dihydroorotase, (4) dihydroorotate dehydrogenase, (5)- (6) UMP synthase (orotate phosphoribosyltransferase plus orotidine-5'-phosphate decarboxylase), (7) UMP kinase, (8) nucleoside diphosphate kinase, (9) CTP synthetase. Source: doi: 10.1199/tab.0018  3. In positive allosteric control of the feedback type, a metabolite formed in early stages of the metabolic chain is a positive regulator in later stages. Such an example can be given with fructose-1,6-bisphosphate, which is an allosteric activator of pyruvate kinase in the glycolytic chain. Glyceraldehyde-3-phosphate Phosphoenolpyruvate Fructose-1,6- Pyruvate diphosphate