Enzyme Inhibition

Enzyme Inhibition galore(postnominal) drugs exert their action by prohibition era of an enzyme action at law in the body. If the activity of an enzyme is vital to the cell or organism, because inhibition whitethorn lead to death of the cell or organism. It is now possible to design new drugs which atomic number 18 enzyme inhibitors once a target enzyme has been identified. Types of Inhibitors A) rechargeable 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 confused. E + I ? EI B) Irreversible InhibitorsThese inhibitors bind real tightly to the enzyme, sometimes by formation of covalent bonds to form an enzyme inhibitor compound rather than a unloose complex. The effect is therefore progressive with time reaching a upper limit when all of the enzyme has responded. This is no t easily turn by simple strong-arm treatments such as dialysis. E + I EI Reversible Inhibition of EnzymesThere atomic number 18 three causes of reversible enzyme inhibitioncompetitive, non-competitive(to a fault called mixed)and uncompetitive. Competitive- counterspyecules which closely resemble the subst roam in size, shape and charge distribution may also slip into the nimble site. This may result in response i. e. the jiffy molecule is some other substrate for the enzyme, or it may result in inhibition because the progressive site is blocked. The inhibitor has a separate equilibrium with the enzyme. The fertilization of substrate and inhibitor is mutually exclusive. E + S ? ES E + P, E + I ?EI all(prenominal) of these equilibria is characterised by a dissociation constant. The first by Km (the Michaelis constant) and the second by Ki which characterises the binding mingled with enzyme and inhibitor. If sufficient S is present then eventually the inhibition by I provideing be overcome. This is the symptomatic test for this type of inhibition. Both I and S compete for the functional enzyme. The activity of an enzyme is described by the following equation (Michaelis- Menton equation) In the front of acompetitive reversible inhibitor, this equation becomesSo theMichaelis constant(which is a reciprocal measure of resemblance 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 amidst E and I. Most importantly,Vmax is unchanged this is diagnostic for this type of inhibition. Ki is exceed defined as the concentration of inhibitor need to slow the reaction to half(a) 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, but theeasiest way to calculate Kiis fr om theratio of the intercepts on the x axis. Without inhibitor the intercept is -1//Km, with inhibitor 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 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 underagely. 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 Reversible gist that the timescale of the inhibition is confusable to that of the enzyme action, usually measured over a a couple of(prenominal) minutes. Irreversiblemeans that the enzyme activity is inhibited for times significantly lengthy than the assay times for the enzyme. It does not necessarily mean that the inhibition volition not reverse given sufficient time i. . hours, years or weeks. nearly of the most interesting examples of enzyme inhibitors as drugs be those which fall between the two extremes and argon sometimes defined as Quasi-Irreversible. These acknowledge tight-binding inhibitors, passage accede line of latitudes and slowly dissociating intermediates. near-Binding inhibitorsandTransition State Analoguesform high affinity complexes with the enzyme and may convey Ki values in the order of nano bomber (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 cause 50% inhibition (I50) is used as a measure of Ki. easy Dissociating Intermediatesreact with the enzyme to form covalent intermediates which take time to dissociate from the enzyme. A Classification of Enzyme Inhibitors as Drugs For a compound to survey as a drug in vivo it will ideally have TWO very important properties. These are PotencyTo work in vivo as an enzyme inhibitor the inhibitor will need to be sloshed seemly so that the dose required is in the order of milligrams to grams.SpecificityIf a compound is a nonspecific enzyme inhibitor it is more than likely to be hepato cyanogenic and exhibit serious side effects. It may be a poison. 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 good reasons, the Km values for enzymes straggle 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 inh ibition will be self-limiting. If an enzyme is not rate limiting, it may be necessary to achieve 90% inhibition ahead any increase in substrate concentration occurs. To do this the inhibitor concentration needs to be approximately 20 times the Ki value. Conformationally certified Competitive Inhibitors- It is possible that a reversible competitive inhibitor which is a conformationally dependent analogue of the substrate will have a much higher(prenominal) affinity for the enzyme han does the substrate and hence can be potent enough to work in vivo at reasonable concentrations. Such compounds may have Ki values in the neighborhood 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 restrict and/or have many non-covalent interactions leading to long abiding complexes. Therefore binding is very tight (Ki in order of 10-9mol L-1to 10-10mol L-1) and these compounds are pot ent enough to act as drugs in vivo.Transition State Analogues- Theoretically, an analogue of a transition state (or reaction intermediate) for the enzyme catalysed reaction will bind much tighter than an analogue of the substrate. The outcome 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 HIV protease and there are now a major(ip) 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 metallic element 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 electron rich will form co-ordinate covalent bonds with these metals will inactivate these proteins. These bonds are strong 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 maneuver chain by ligand formation with the Cu atom at the total of its mechanism. Similarly, carbon monoxide complexes with the Fe atom in the haem cofactor of haemoglobi n. b. Heavy metal ions e. g. mercury, lead etc. These are common irreversible inhibitors because of their ability to complex firmly with particular groups in enzymes. These effects can be reversed by treatment with chelating agents such as EDTA (ethylene di-amino tetra acetic caustic). 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 rapidly 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 remember these enzymes as part of the link reaction and the citric acid cycle. When these enzymes are blocke d, ventilation system stops. Arsenic derivatives have been prepared as very poisonous struggle gases e. g. Lewisite. antidote calledDimercaprol (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 constant compound and thus stopping it from blocking the thiol groups in lipoic acid. Dimercaprolis used these days as an antidote to toxic condition with heavy 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 excited 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 efficacio us in elucidating enzyme mechanisms but their reactive nature makes them likely to be toxic when used in vivo.Mechanism-based Inhibitors (suicide reagents) The principle of this sort of inhibition is that a pseudo substrate is accepted by the enzyme which then catalyses the production of its give inhibitor which reacts covalently in the active site. Such inhibitors should be specific as strong 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, wizard inhibitors Tight binding, HIV protease inhibitors Transition state analogues, Neostigmine, Penicillin Slowly dissociating intermediates DFP Irreversible group specific reagent, Clavulanate mechanism-based irreversible i nhibitor. 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 fit 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 below 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 classify reagents e. g. Arsenic (III), Iodoacetamide DFP action on esterases Affinity labels TPCK on Chymotrypsin Mechanism found (suicide inhibitors) e. g. Clavulanate onlactamase Enzyme Inhibito rs as Drugs ENZYME INHIBITOR(S) USES Acetylcholinesterase Edrophonium Neostigmine Eserine Myasthenia Gravis Glaucoma Paralytic Ileus Monoamine Oxidase Tranylcypramine printing Xanthine Oxidase Allopurinol Gout, adjunct to pubic louse chemotherapy Carbonic Anhydrase Acetazolamide Diuresis Dihydrofolate Reductase Methotrexate LeukaemiaTranspeptidase Penicillin Antibacterial Cyclo-oxygenase acetylsalicylic acid 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 philia Disease Phospodiesterase V Viagra Erectile dysfunction