Enzyme Inhibition

Enzyme banning Many drugs exert their coifion by curtailment of an enzyme activity in the body. If the activity of an enzyme is vital to the booth or organism, and then stifling may lead to death of the cell or organism. It is now executable to design new drugs which be enzyme inhibitors once a target enzyme has been identified. Types of Inhibitors A) Reversible InhibitorsThe effect of the inhibitor is instantaneous, and it can be removed from the enzyme by dialysis so that the enzyme activity is returned to normal.Such inhibitors interact with the enzyme by weak non-covalent bonds to form an enzyme inhibitor complex. E + I ? EI B) Irreversible InhibitorsThese inhibitors halt very(prenominal) tightly to the enzyme, some epochs by formation of covalent bonds to form an enzyme inhibitor deepen rather than a loose complex. The effect is therefore progressive with time reaching a maximal when all of the enzyme has reacted. This is not easily reversed by uncomplicated physica l treatments such as dialysis. E + I EI Reversible Inhibition of EnzymesThere are three types of reversible enzyme forbiddingmilitant, non-competitive( as well called mixed)and uncompetitive. Competitive- moleecules which closely resemble the substrate in size, shape and charge distri providedion may also slip into the active grade. This may resolving power in reaction i. e. the second molecule is another substrate for the enzyme, or it may result in inhibition because the active site is blocked. The inhibitor has a separate equilibrium with the enzyme. The binding of substrate and inhibitor is mutually exclusive. E + S ? ES E + P, E + I ?EI Each of these equilibria is characterised by a dissociation regular. The startle by Km (the Michaelis constant) and the second by Ki which characterises the binding between enzyme and inhibitor. If sufficient S is present then eventually the inhibition by I bequeath be overcome. This is the diagnostic test for this type of inhibition. twain I and S compete for the available enzyme. The activity of an enzyme is described by the following equation (Michaelis- Menton equation) In the presence of acompetitive reversible inhibitor, this equation becomesSo theMichaelis constant(which is a reciprocal flier of semblance of E and S) is changed by the factor 1 + I/Ki where I is the inhibitor concentration andKi is the dissociation constant for the equilibrium between E and I. Most importantly,Vmax is unchanged this is diagnostic for this type of inhibition. Ki is best be as the concentration of inhibitor required to slow the reaction to half the rate it shows in the absence of inhibitor. It is a reciprocal measure of the affinity of E and I. Lineweaver-Burk Plot for Competitive Reversible InhibitionTheintercept on the y axis represents 1/Vmax. The slope is altered by the factor 1 + I/Ki, except theeasiest way to play Kiis from theratio of the intercepts on the x axis. Without inhibitor the intercept is -1//Km, with i nhibitor it is -1/Km(1+I/Ki), so the ratio (bigger over smaller so it is greater than 1) is 1 + I/Ki. Easiest way to calculate Kiis from theratio of the intercepts on the x axis. Equation other(a) Types of Reversible Inhibition Uncompetitive- This type of reversible inhibition is said to occur when the inhibitor binds with the enzyme-substrate complex rather than the enzyme.Substrate and inhibitor bind dependently. Noncompetitive (Mixed)- This type occurs when the inhibitor binds to both the enzyme and enzyme-substrate complex. Substrate and inhibitor bind independently. Irreversible Inhibition of Enzymes Reversiblemeans that the timescale of the inhibition is similar to that of the enzyme action, usually measured over a few minutes. Irreversiblemeans that the enzyme activity is hold for times significantly longer than the assay times for the enzyme. It does not necessarily mean that the inhibition will not reverse given sufficient time i. . hours, days or weeks. Some of the most kindle examples of enzyme inhibitors as drugs are those which fall between the both extremes and are sometimes defined as Quasi-Irreversible. These accept tight-binding inhibitors, transition state linears and slowly dissociating intermediates. Tight-Binding inhibitorsandTransition State Analoguesform high affinity complexes with the enzyme and may move over Ki values in the post of nanomolar (10-9mol L-1). The value of Ki will be very important in describing the potency of this type of inhibitor.As a rough guide the inhibitor concentration causing 50% inhibition (I50) is used as a measure of Ki. Slowly Dissociating Intermediatesreact with the enzyme to form covalent intermediates which deal time to dissociate from the enzyme. A Classification of Enzyme Inhibitors as Drugs For a involved to dally as a drug in vivo it will ideally fox TWO very important properties. These are military capabilityTo work in vivo as an enzyme inhibitor the inhibitor will need to be unshakable enough so that the process required is in the order of milligrams to grams.SpecificityIf a compound is a nonspecific enzyme inhibitor it is more likely to be toxic and exhibit serious side effects. It may be a toxicant. Simple Reversible- A simple reversible inhibitor binds to the enzyme and decreases the enzyme activity instantaneously and reverses within the time of the enzyme action. The inhibitor binds non-covalently (ionic interactions, hydrogen bonds, Van Der Waals forces) to the enzyme and the strength of binding is of a similar order to the substrate i. e. Ki will be of similar size to Km. For very erect reasons, the Km values for enzymes vary between about 10-2mol L-1to 10-6mol L-1.Unlikely to be potent enough to work in vivo where competition occurs in a dynamic metabolic situation. For a simple competitive inhibitor the inhibition will be self-limiting. If an enzyme is not rate limiting, it may be necessary to achieve 90% inhibition before any increase in substrate con centration occurs. To do this the inhibitor concentration needs to be approximately 20 times the Ki value. Conformationally Restricted Competitive Inhibitors- It is possible that a reversible competitive inhibitor which is a conformationally cut back analogue of the substrate will beget a much higher affinity for the enzyme han does the substrate and hence can be potent enough to work in vivo at tenable concentrations. Such compounds may have Ki values in the region of 1 x 10-7mol L-1 Quasi-Irreversible Tight Binding Inhibitors- This is an extension of the previous class i. e. competitive inhibitors which are conformationally restricted and/or have many non-covalent interactions leading to long lasting complexes. Therefore binding is very tight (Ki in order of 10-9mol L-1to 10-10mol L-1) and these compounds are potent enough to act as drugs in vivo.Transition State Analogues- Theoretically, an analogue of a transition state (or reaction intermediate) for the enzyme catalysed reac tion will bind much tighter than an analogue of the substrate. The issuance is a potent and potentially specific inhibitor. Theoretically, Ki values can be very low. In practice if Ki values in the region of Nano molar can be achieved, these are potent enough to work in vivo. As we shall see, there has been much work in this area on proteases including human immunodeficiency virus protease and there are now a major class of drugs which has been developed on this principle.Slowly Dissociating Intermediates- Some enzymes form covalent intermediates as part of their mechanism e. g. acetylcholinesterase. It is possible for a compound to act as a pseudo-substrate and be converted into a long lasting intermediate. Such an inhibition is time dependent and in some cases is virtually irreversible. Sometimes the intermediate is hydrolysed in minutes or hours but this is still much longer than the normal enzyme mechanism when the intermediate would last only milliseconds. Examples include the anticholinesterases neostigmine and physostigmine (eserine) and penicillin.Irreversible Nonspecific a. Heavy admixture poisons e. g. cyanide, hydrogen sulphide, carbon monoxide- Some enzymes and other important proteins such as Haemoglobin and Cytochromes, require metals as cofactors. These metals are often transition metals such as Fe, Cu, Mn, Zn and ligands which are negatron rich will form co-ordinate covalent bonds with these metals will inactivate these proteins. These bonds are difficult and very often these ligands are toxic because of this irreversible inactivation.Cyanide reacts with cytochrome oxidase which is the terminal electron carrier in the electron transport chain by ligand formation with the Cu atom at the centre of its mechanism. Similarly, carbon monoxide complexes with the Fe atom in the haem cofactor of haemoglobin. b. Heavy metal ions e. g. mercury, lead and so on These are common irreversible inhibitors because of their ability to complex firmly with pa rticular chemical groups in enzymes. These effects can be reversed by treatment with chelating agents such as EDTA (ethylene di-amino tetra acetic venomous). c. Thiol poisons e. . alkylating agents, Arsenic (III) Many enzymes contain thiol (-SH) groups in amino acid side chains cysteine, which are essential for catalytic activity. Any compound which reacts with these functional groups will poison the enzyme. E. g. Iodoacetamide(alkylating agent) Arsenic- The most toxic form of Arsenic is As (III) as in arsenite AsO2. In this form, Arsenic reacts quickly withthiol groups, especially with dithiols such as lipoic acid which is an essential cofactor for some important enzymes such as pyruvate dehydrogenase and -ketoglutarate dehyrdrogenase.You should record these enzymes as part of the link reaction and the citric acid cycle. When these enzymes are blocked, respiration stops. Arsenic derivatives have been prepared as very poisonous war gases e. g. Lewisite. antidote calledDimercapr ol (British Anti-Lewisite)was designed by incorporating two thiols for the poison to react with. The two thiol groups react with the arsenical war gas forming a stable compound and thus stopping it from blocking the thiol groups in lipoic acid. Dimercaprolis used these days as an antidote to intoxication with gruelling metals such as antimony, arsenic, mercury, bismuth, gold, thallium.It is also used in conjunction with pencillamine in the treatment of lead poisoning (see BNF). Specific Irreversible Inhibitors Affinity Labels (Active site directed irreversible inhibitors)- An analogue of the substrate which binds to the active site of an enzyme, but which contains a chemically reactive group, has the potential to form covalent bonds with side chains at or near the active site. These inhibitors are irreversible and have been very useful in elucidating enzyme mechanisms but their reactive nature makes them likely to be toxic when used in vivo.Mechanism-based Inhibitors (suicide reag ents) The principle of this chassis of inhibition is that a pseudo substrate is accepted by the enzyme which then catalyses the production of its own inhibitor which reacts covalently in the active site. Such inhibitors should be specific as well as potent. Certain monoamine oxidase inhibitors have this mechanism, also the -lactamase inhibitors (e. g. clavulanate). The pyridoxal phosphate (vitamin B6) dependent enzymes have been a particular candidate for the development of this kind of inhibitor (e. g. difluoromethyldopa). Enzyme inhibitorsEdrophonium conformationally restricted competitive reversible, ACE inhibitors Tight binding, HIV protease inhibitors Transition state analogues, Neostigmine, Penicillin Slowly dissociating intermediates DFP Irreversible group specific reagent, Clavulanate mechanism-based irreversible inhibitor. Types of Enzyme Inhibitors Simple Reversible Competitive (also uncompetitive, noncompetitive, mixed) Simple substrate analogues Michaelis-Menten kinetics Ki in region of Km i. e. 10-2 10-6M Restricted Conformation Rigid shape similar to favoured substrate check off Ki less than Km e. g. drophonium as inhibitor of acetylcholinesterase Quasi-Irreversible Tight Binding Ki can be in region of nanomolar E. g. ACE inhibitors Captopril, enalapril etc. Transition State Analogues. Binding constant theoretically infra nanomolar Inhibitors of proteinases e. g. pepsin, renin, HIV proteinase Slowly Dissociating Intermediates time dependent kinetics e. g. neostigmine, eserine as anticholinesterases Penicillin Irreversible Heavy metal poisons etc Cyanide, Hydrogen Sulphide, Carbon Monoxide Group reagents e. g. Arsenic (III), Iodoacetamide DFP action on esterases Affinity labels TPCK on Chymotrypsin Mechanism establish (suicide inhibitors) e. g. Clavulanate onlactamase Enzyme Inhibitors as Drugs ENZYME INHIBITOR(S) USES Acetylcholinesterase Edrophonium Neostigmine Eserine Myasthenia Gravis Glaucoma Paralytic Ileus Monoamine Oxidas e Tranylcypramine Depression Xanthine Oxidase Allopurinol Gout, adjunct to crabby person chemotherapy Carbonic Anhydrase Acetazolamide Diuresis Dihydrofolate Reductase Methotrexate LeukaemiaTranspeptidase Penicillin Antibacterial Cyclo-oxygenase Aspirin etc. Non-steroidal anti-inflammatory drugs Analgesia Anti-inflammatory Anti-platelet Angiotensin Converting Enzyme (ACE) Captopril, enalapril, lisinopril etc. Anti-hypertension Thymidylate Synthetase Fluorouracil Cancer chemotherapy Penicillinase (-lactamase) Clavulanate etc Anti-bacterial HIV proteinase Saquinovar etc HIV treatment Reverse Transcriptase AZT HIV treatment HMG-CoA Reductase Statins, pravastatin etc. Coronary Heart Disease Phospodiesterase V Viagra Erectile dysfunction