Enzyme Inhibition: Types and Reversibility

Enzyme Inhibition Many drugs exert their action by inhibition of an enzyme activity in the body. If the activity of an enzyme is vital to the cell or organism, then inhibition may lead to death of the cell or organism. It is now possible to design new drugs which are enzyme inhibitors once a target enzyme has been identified. Types of Inhibitors A) Reversible Inhibitors: The 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 Inhibitors: These inhibitors bind very tightly to the enzyme, sometimes by formation of covalent bonds to form an enzyme inhibitor compound rather than a loose complex. The effect is therefore progressive with time reaching a maximum when all of the enzyme has reacted. This is not easily reversed by simple physical treatments such as dialysis. E + I > EI Reversible Inhibition of Enzymes

There are three types of reversible enzyme inhibition; competitive, non-competitive (also called mixed) and uncompetitive. Competitive- molecules which closely resemble the substrate in size, shape and charge distribution may also slip into the active site. This may result 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 ?

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EI Each 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 between enzyme and inhibitor. If sufficient [S] is present then eventually the inhibition by I will be overcome. This is the diagnostic test for this type of inhibition. Both 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 a competitive reversible inhibitor, this equation becomes;

So the Michaelis constant (which is a reciprocal measure of affinity of E and S) is changed by the factor 1 + [I]/Ki where [I] is the inhibitor concentration and Ki 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 defined 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 Inhibition

The intercept on the y axis represents 1/Vmax. The slope is altered by the factor 1 + [I]/Ki, but the easiest way to calculate Ki is from the ratio 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 Ki is from the ratio 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 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 Reversible means that the timescale of the inhibition is similar to that of the enzyme action, usually measured over a few minutes. Irreversible means that the enzyme activity is inhibited 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 interesting examples of enzyme inhibitors as drugs are those which fall between the two extremes and are sometimes defined as Quasi-Irreversible. These include tight-binding inhibitors, transition state analogues and slowly dissociating intermediates. Tight-Binding inhibitors and Transition State Analogues form high affinity complexes with the enzyme and may have Ki values in the order of nanomolar (10-9 mol 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 Intermediates react 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 work as a drug in vivo it will ideally have TWO very important properties. These are; Potency To work in vivo as an enzyme inhibitor the inhibitor will need to be potent enough so that the dose required is in the order of milligrams to grams.

Specificity If a compound is a nonspecific enzyme inhibitor it is more likely to be toxic 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 Waal's 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 vary between about 10-2 mol L-1 to 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 concentration 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 restricted analogue of the substrate will have a much higher 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 region of 1 x 10-7 mol 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 10-9 mol L-1 to 10-10 mol 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 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 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 metal 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 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 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 acid). 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 with thiol 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 blocked, respiration stops. Arsenic derivatives have been prepared as very poisonous war gases e. g. Lewisite. antidote called Dimercaprol ('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. Dimercaprol is used these days as an antidote to poisoning 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 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 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 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 inhibitors:

Edrophonium - 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 - 10-6 M| 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| | Group reagents| e. g. Arsenic (III), Iodoacetamide| | DFP action on esterases| | Affinity labels| TPCK on Chymotrypsin| | Mechanism Based ('suicide inhibitors')| e. g. Clavulanate onlactamase| Enzyme Inhibitors as Drugs ENZYME| INHIBITOR(S)| USES| Acetylcholinesterase| Edrophonium Neostigmine Eserine| Myasthenia Gravis Glaucoma Paralytic Ileus| Monoamine Oxidase| Tranylcypramine| Depression| Xanthine Oxidase| Allopurinol| Gout, adjunct to Cancer chemotherapy| Carbonic Anhydrase| Acetazolamide| Diuresis| Dihydrofolate Reductase| Methotrexate| Leukaemia|

Transpeptidase| 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|

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