View source for Enzyme inhibitor

{{Short description|Molecule that blocks enzyme activity}}
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[[File:Enzyme inhibitors 2.svg|thumb|alt=cartoon depiction of an enzyme binding substrate to its active site and releasing product (top), and an inhibitor binding to the active site, thus preventing substrate binding|Top: [[enzyme]] (E) accelerates conversion of [[Substrate (biochemistry)|substrates]] (S) to [[Product (chemistry)|products]] (P). Bottom: by binding to the enzyme, inhibitor (I) blocks binding of substrate. Binding site shown in blue checkerboard, substrate as black rectangle, and inhibitor as green rounded rectangle.]]

An '''enzyme inhibitor''' is a [[molecule]] that binds to an [[enzyme]] and blocks its [[Enzyme activity|activity]]. Enzymes are [[protein]]s that speed up [[chemical reaction]]s necessary for [[life]], in which [[Substrate (biochemistry)|substrate molecules]] are converted into [[Product (chemistry)|product]]s.<ref name = "Copeland_2013">{{cite book | vauthors = Copeland RA | chapter = Why Enzymes as Drug Targets? Enzyme are Essential for Life | pages = 1–23 |title=Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide for Medicinal Chemists and Pharmacologists |date=March 2013 |publisher=John Wiley & Sons, Inc. |isbn=978-1-118-48813-3 | doi = 10.1002/9781118540398.ch1 |edition=Second}}</ref> An enzyme [[Enzyme catalysis|facilitates]] a specific chemical reaction by binding the substrate to its [[active site]], a specialized area on the enzyme that accelerates the [[Rate-determining step|most difficult step of the reaction]].

An enzyme inhibitor stops ("inhibits") this process, either by binding to the enzyme's active site (thus preventing the substrate itself from binding) or by binding to another site on the enzyme such that the enzyme's [[catalysis]] of the reaction is blocked. Enzyme inhibitors may bind [[Reversible reaction|reversibly]] or irreversibly. Irreversible inhibitors form a [[Covalent bond|chemical bond]] with the enzyme such that the enzyme is inhibited until the chemical bond is broken. By contrast, reversible inhibitors bind [[Non-covalent interactions|non-covalently]] and may spontaneously leave the enzyme, allowing the enzyme to resume its function. Reversible inhibitors produce different types of inhibition depending on whether they bind to the enzyme, the enzyme-substrate complex, or both.

Enzyme inhibitors play an important role in all cells, since they are generally specific to one enzyme each and serve to control that enzyme's activity. For example, enzymes in a [[metabolic pathway]] may be inhibited by molecules produced later in the pathway, thus curtailing the production of molecules that are no longer needed. This type of [[negative feedback]] is an important way to maintain [[Homeostasis|balance]] in a [[Cell (biology)|cell]].<ref name="Sauro_2017" /> Enzyme inhibitors also control essential enzymes such as [[protease]]s or [[nuclease]]s that, if left unchecked, may damage a cell. Many [[poison]]s produced by animals or plants are enzyme inhibitors that block the activity of crucial enzymes in prey or [[predator]]s.

Many [[Medication|drug molecules]] are enzyme inhibitors that inhibit an aberrant human enzyme or an enzyme critical for the survival of a [[pathogen]] such as a [[virus]], [[bacterium]] or [[parasite]]. Examples include [[methotrexate]] (used in [[chemotherapy]] and in treating [[rheumatic arthritis]]) and the [[protease inhibitors]] used to treat [[HIV/AIDS]]. Since anti-pathogen inhibitors generally target only one enzyme, such drugs are highly [[Specificity (biochemistry)|specific]] and generally produce few side effects in humans, provided that no [[Sequence homology|analogous]] enzyme is found in humans. (This is often the case, since such [[pathogen]]s and [[human]]s are [[Genetic distance|genetically distant]].) Medicinal enzyme inhibitors often have low [[dissociation constant]]s, meaning that only a minute amount of the inhibitor is required to inhibit the enzyme. A low concentration of the enzyme inhibitor reduces the risk for [[Hepatotoxicity|liver]] and [[Nephrotoxicity|kidney damage]] and other [[adverse drug reaction]]s in humans. Hence the discovery and refinement of enzyme inhibitors is an active area of research in [[biochemistry]] and [[pharmacology]].

==Structural classes==
Enzyme inhibitors are a chemically diverse set of substances that range in size from organic [[small molecule]]s to macromolecular [[protein]]s.

Small molecule inhibitors include essential [[primary metabolite]]s that inhibit upstream enzymes that produce those metabolites. This provides a negative feedback loop that prevents over production of metabolites and thus maintains cellular [[homeostasis]] (steady internal conditions).<ref name = "Plaxton_2004" /><ref name="Sauro_2017" /> Small molecule enzyme inhibitors also include [[secondary metabolite]]s, which are not essential to the organism that produces them, but provide the organism with an evolutionary advantage, in that they can be used to repel predators or competing organisms or immobilize prey.<ref name="Haefner_2003" /> In addition, many drugs are small molecule enzyme inhibitors that target either disease-modifying enzymes in the patient<ref name = "Copeland_2013" />{{rp|5}} or enzymes in pathogens which are required for the growth and reproduction of the pathogen.<ref name = "Gualerzi_2013">{{cite book | vauthors = Gualerzi CO, Brandi L, Fabbretti A, Pon CL |title=Antibiotics: Targets, Mechanisms and Resistance. |date=2013 |publisher=John Wiley & Sons |location=Hoboken |isbn=978-3-527-65970-8}}</ref>

In addition to small molecules, some proteins act as enzyme inhibitors. The most prominent example are [[serpin]]s ('''ser'''ine '''p'''rotease '''in'''hibitors) which are produced by animals to protect against inappropriate enzyme activation and by plants to prevent predation.<ref name="Sanrattana_2019">{{cite journal | vauthors = Sanrattana W, Maas C, de Maat S | title = SERPINs-From Trap to Treatment | journal = Frontiers in Medicine | volume = 6 | issue = | pages = 25 | date = 2019 | pmid = 30809526 | pmc = 6379291 | doi = 10.3389/fmed.2019.00025 | doi-access = free }}</ref> Another class of inhibitor proteins is the [[ribonuclease inhibitor]]s, which bind to [[ribonuclease]]s in one of the tightest known [[protein–protein interaction]]s.<ref>{{cite journal | vauthors = Shapiro R, Vallee BL | title = Interaction of human placental ribonuclease with placental ribonuclease inhibitor | journal = Biochemistry | volume = 30 | issue = 8 | pages = 2246–2255 | date = February 1991 | pmid = 1998683 | doi = 10.1021/bi00222a030 }}</ref> A special case of protein enzyme inhibitors are [[zymogen]]s that contain an autoinhibitory [[N-terminal]] peptide that binds to the active site of enzyme that [[Intramolecular reaction|intramolecularly]] blocks its activity as a protective mechanism against uncontrolled catalysis. The N{{nbh}}terminal peptide is cleaved (split) from the zymogen enzyme precursor by another enzyme to release an active enzyme.<ref name="Boon_2020">{{cite journal | vauthors = Boon L, Ugarte-Berzal E, Vandooren J, Opdenakker G | title = Protease propeptide structures, mechanisms of activation, and functions | journal = Critical Reviews in Biochemistry and Molecular Biology | volume = 55 | issue = 2 | pages = 111–165 | date = April 2020 | pmid = 32290726 | doi = 10.1080/10409238.2020.1742090 | s2cid = 215772580 }}</ref>

The [[binding site]] of inhibitors on enzymes is most commonly the same site that binds the [[Substrate (biochemistry)|substrate]] of the enzyme. These [[active site]] inhibitors are known as [[Allosteric regulation#Allosteric sites as drug targets|orthosteric]] ("regular" orientation) inhibitors.<ref name = "Rydzewski_2010">{{cite book |vauthors=Rydzewski RM |chapter=Chapter 7.2.1: Competition and Allostery |title=Real World Drug Discovery: A Chemist's Guide to Biotech and Pharmaceutical Research |publisher=Elsevier |location=Amsterdam |isbn=978-0-08-091488-6 |edition=1st |chapter-url=https://books.google.com/books?id=9ELvBMCyKKwC&pg=PA288 |pages=281–285 |date=2010 |access-date=20 July 2022 |archive-date=28 March 2023 |archive-url=https://web.archive.org/web/20230328005931/https://books.google.com/books?id=9ELvBMCyKKwC&pg=PA288 |url-status=live }}</ref> The mechanism of orthosteric inhibition is simply to prevent substrate binding to the enzyme through direct competition which in turn prevents the enzyme from catalysing the conversion of substrates into products. Alternatively, the inhibitor can bind to a site remote from the enzyme active site. These are known as [[Allosteric inhibitor|allosteric]] ("alternative" orientation) inhibitors.<ref name = "Rydzewski_2010" /> The mechanisms of allosteric inhibition are varied and include changing the [[Conformational isomerism|conformation]] (shape) of the enzyme such that it can no longer bind substrate ([[Enzyme kinetics|kinetically]] indistinguishable from competitive orthosteric inhibition)<ref name="pmid30917186">{{cite journal | vauthors = Jakubík J, Randáková A, El-Fakahany EE, Doležal V | title = Analysis of equilibrium binding of an orthosteric tracer and two allosteric modulators | journal = PLOS ONE | volume = 14 | issue = 3 | pages = e0214255 | date = 2019 | pmid = 30917186 | pmc = 6436737 | doi = 10.1371/journal.pone.0214255 | bibcode = 2019PLoSO..1414255J | doi-access = free }}</ref> or alternatively stabilise binding of substrate to the enzyme but lock the enzyme in a conformation which is no longer catalytically active.<ref name = "Patrick_2013">{{cite book |vauthors=Patrick GL |chapter=Chapter 7: Enzymes as Drug Targets |title=An Introduction to Medicinal Chemistry |date=2013 |publisher=Oxford University Press |location=Oxford, UK |isbn=978-0-19-969739-7 |page=90 |edition=Fifth |chapter-url=https://books.google.com/books?id=Pj7xJRuhZxUC&dq=mechanism+of+allosteric+inhibition+enzyme+catalysis+competitive+noncompetitive&pg=PA90 |access-date=20 July 2022 |archive-date=20 July 2022 |archive-url=https://web.archive.org/web/20220720171418/https://books.google.com/books?id=Pj7xJRuhZxUC&dq=mechanism+of+allosteric+inhibition+enzyme+catalysis+competitive+noncompetitive&pg=PA90 |url-status=live }}</ref>

==Reversible inhibitors==
{{multiple image
| header      = Inhibition mechanism schematic
| image1      = Inhibition mechanism (reversible).svg
| image2      = Inhibition schematic (reversible).svg
| alt1        = chemical equilibrium reaction formula for competitive, uncompetitive, non-competitive, and mixed inhibition
| alt2        = schematic diagram of the three types of reversible inhibitors
| caption1    = Kinetic mechanisms for reversible inhibition. Substrate (S) binding to enzyme (E) in blue, catalysis releasing product (P) in red, inhibitor (I) binding to enzyme in green.
| caption2    = Schematics for reversible inhibition. Binding site in blue, substrate in black, inhibitor in green, and allosteric site in light green.
| footer      = Competitive inhibitors usually bind to the active site. Non-competitive bind to a remote (allosteric) site. Uncompetitive inhibitors only bind once the substrate is bound, fully disrupting catalysis, and mixed inhibition is similar but with only partial disruption of catalysis.
}}

Reversible inhibitors attach to enzymes with [[non-covalent interaction]]s such as [[hydrogen bond]]s, [[hydrophobic interaction]]s and [[ionic bond]]s.<ref>{{cite book |vauthors=Kuriyan J, Konforti B, Wemmer D |title=The Molecules of Life : Physical and Chemical Principles |date=2012 |publisher=Garland Science |location=Boca Raton, FL |isbn=978-1-135-08892-7 |chapter=Molecular Recognition: The Thermodynamics of Binding |chapter-url=https://books.google.com/books?id=jwcPBAAAQBAJ&pg=PA531 |pages=531–580 |edition=First |access-date=7 June 2022 |archive-date=7 June 2022 |archive-url=https://web.archive.org/web/20220607154842/https://books.google.com/books?id=jwcPBAAAQBAJ&pg=PA531 |url-status=live }}</ref> Multiple weak bonds between the inhibitor and the enzyme [[active site]] combine to produce strong and specific binding.

In contrast to irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or [[Kidney dialysis|dialysis]]. A special case is '''covalent reversible inhibitors''' that form a chemical bond with the enzyme, but the bond can be cleaved so the inhibition is fully reversible.<ref name="Tuley_2018">{{cite journal | vauthors = Tuley A, Fast W | title = The Taxonomy of Covalent Inhibitors | journal = Biochemistry | volume = 57 | issue = 24 | pages = 3326–3337 | date = June 2018 | pmid = 29689165 | pmc = 6016374 | doi = 10.1021/acs.biochem.8b00315 }}</ref>

Reversible inhibitors are generally categorized into four types, as introduced by [[W.&nbsp;Wallace Cleland|Cleland]] in 1963.<ref name="Cleland_1963">{{cite journal |vauthors=Cleland WW |date=February 1963 |title=The kinetics of enzyme-catalysed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory |journal=Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects |volume=67 |pages=173–187 |doi=10.1016/0926-6569(63)90226-8 |pmid=14021668}}</ref> They are classified according to the effect of the inhibitor on the ''V<sub>max</sub>'' (maximum reaction rate catalysed by the enzyme) and ''K<sub>m</sub>'' (the concentration of substrate resulting in half maximal enzyme activity) as the concentration of the enzyme's substrate is varied.<ref>{{cite book | vauthors = Berg J, Tymoczko J, Stryer L | date = 2002 | url = https://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=stryer.TOC&depth=2 | title = Biochemistry | publisher = W. H. Freeman and Company | isbn = 978-0-7167-4955-4 | access-date = 31 August 2017 | archive-date = 26 September 2009 | archive-url = https://web.archive.org/web/20090926163540/http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=bv.View..ShowTOC&rid=stryer.TOC&depth=2 | url-status = live }}</ref><ref name="NIH_CTT">{{cite web |title=Types of Inhibition |url= https://assay.nih.gov/assay/index.php/Types_of_Inhibition |publisher=NIH Center for Translational Therapeutics |access-date=2 April 2012 |url-status=dead |archive-url=https://web.archive.org/web/20110908030859/http://assay.nih.gov/assay/index.php/Types_of_Inhibition |archive-date=8 September 2011 }}</ref>

===Competitive===
In [[competitive inhibition]] the substrate and inhibitor cannot bind to the enzyme at the same time.<ref name = "Cornish-Bowden_2012">{{cite book | vauthors = Cornish-Bowden A |title=Fundamentals of Enzyme Kinetics |date=2012 |publisher=Wiley-VCH |location=Weinheim |isbn=978-3-527-66549-5 |edition=4th}}</ref>{{rp|134}} This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor ''compete'' for access to the enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate (''V<sub>max</sub>'' remains constant), i.e., by out-competing the inhibitor.<ref name = "Cornish-Bowden_2012" />{{rp|134–135}} However, the apparent ''K<sub>m</sub>'' will increase as it takes a higher concentration of the substrate to reach the ''K<sub>m</sub>'' point, or half the ''V<sub>max</sub>''. Competitive inhibitors are often similar in structure to the real substrate (see for example the "methotrexate versus folate" figure in the [[#Drugs|"Drugs" section]]).<ref name = "Cornish-Bowden_2012" />{{rp|134}}

===Uncompetitive===
In [[uncompetitive inhibition]] the inhibitor binds only to the enzyme-substrate complex.<ref name = "Cornish-Bowden_2012" />{{rp|139}} This type of inhibition causes ''V<sub>max</sub>'' to decrease (maximum velocity decreases as a result of removing activated complex) and ''K<sub>m</sub>'' to decrease (due to better binding efficiency as a result of [[Le Chatelier's principle]] and the effective elimination of the ES complex thus decreasing the ''K<sub>m</sub>'' which indicates a higher binding affinity).<ref name=Segel/> Uncompetitive inhibition is rare.<ref name = "Cornish-Bowden_2012" />{{rp|139}}<ref name = "Palmer_2007">{{cite book | vauthors = Palmer T, Bonner PL | chapter = Enzyme inhibition. | title = Enzymes: Biochemistry, Biotechnology, Clinical Chemistry | edition = 2nd | date = 2007 | doi = 10.1533/9780857099921.2.126 | pages = 126–152 (135) | isbn = 978-1-904275-27-5 | publisher = Woodhead Publishing }}</ref>

===Non-competitive===
In [[non-competitive inhibition]] the binding of the inhibitor to the enzyme reduces its [[Enzyme activity|activity]] but does not affect the binding of substrate.<ref name="NIH_CTT" /> This type of inhibitor binds with equal affinity to the free enzyme as to the enzyme-substrate complex. It can be thought of as having the ability of competitive and uncompetitive inhibitors, but with no preference to either type. As a result, the extent of inhibition depends only on the concentration of the inhibitor. ''V<sub>max</sub>'' will decrease due to the inability for the reaction to proceed as efficiently, but ''K<sub>m</sub>'' will remain the same as the actual binding of the substrate, by definition, will still function properly.<ref name = "Delaune_2021">{{cite book |vauthors=Delaune KP, Alsayouri K |title=StatPearls |chapter=Physiology: Noncompetitive Inhibitor |date=September 2021 |publisher=StatPearls Publishing |location=Treasure Island (FL) |page=31424826 |pmid=31424826 |chapter-url=https://www.ncbi.nlm.nih.gov/books/NBK545242/ |access-date=3 April 2022 |archive-date=28 November 2021 |archive-url=https://web.archive.org/web/20211128175329/https://www.ncbi.nlm.nih.gov/books/NBK545242/ |url-status=live }}</ref>

===Mixed===
In [[mixed inhibition]] the inhibitor may bind to the enzyme whether or not the substrate has already bound. Hence mixed inhibition is a combination of competitive and noncompetitive inhibition.<ref name="NIH_CTT" /> Furthermore, the affinity of the inhibitor for the free enzyme and the enzyme-substrate complex may differ.<ref name = "Cornish-Bowden_2012" />{{rp|136–139}} By increasing concentrations of substrate [S], this type of inhibition can be reduced (due to the competitive contribution), but not entirely overcome (due to the noncompetitive component).<ref name = "Voet_2016" />{{rp|381–382}} Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an [[allosteric]] effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this [[allosteric site]] changes the [[Conformational isomerism|conformation]] (that is, the [[tertiary structure]] or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced.<ref name="Buker_2019">{{cite journal | vauthors = Buker SM, Boriack-Sjodin PA, Copeland RA | title = Enzyme-Inhibitor Interactions and a Simple, Rapid Method for Determining Inhibition Modality | journal = SLAS Discovery: Advancing Life Sciences R & D | volume = 24 | issue = 5 | pages = 515–522 (516) | date = June 2019 | pmid = 30811960 | doi = 10.1177/2472555219829898 | s2cid = 73480979 | quote = In some cases, the inhibitor may bind to a distinct site on the enzyme that is in allosteric communication with the substrate binding pocket. In many cases, allosteric, substrate competitive compounds result in conformational changes to the enzyme that change the ability of the enzyme to bind substrate. | doi-access = free }}</ref>

These four types of inhibition can also be distinguished by the effect of increasing the substrate concentration [S] on the degree of inhibition caused by a given amount of inhibitor. For competitive inhibition the degree of inhibition is reduced by increasing [S], for noncompetitive inhibition the degree of inhibition is unchanged, and for uncompetitive (also called anticompetitive) inhibition the degree of inhibition increases with [S].<ref>{{cite book | vauthors = Laidler KJ |title=Physical Chemistry with Biological Applications |date=1978 |publisher=Benjamin/Cummings |isbn=978-0-8053-5680-9 |page=437}}</ref>

===Quantitative description===
Reversible inhibition can be described quantitatively in terms of the inhibitor's [[Dissociation constant|binding]] to the enzyme and to the enzyme-substrate complex, and its effects on the [[Enzyme kinetics|kinetic constants]] of the enzyme.<ref name = "Bisswanger_2017">{{cite book | vauthors = Bisswanger H |title=Enzyme Kinetics: Principles and Methods. |date=2017 |publisher=John Wiley & Sons, Incorporated |location=Newark |isbn=978-3-527-80647-8 |edition=3rd}}</ref>{{rp|6}} In the classic [[Michaelis-Menten kinetics|Michaelis-Menten]] scheme (shown in the "inhibition mechanism schematic" diagram), an enzyme (E) binds to its substrate (S) to form the enzyme–substrate complex ES. Upon catalysis, this complex breaks down to release product P and free enzyme.<ref name = "Bisswanger_2017" />{{rp|55}} The inhibitor (I) can bind to either E or ES with the [[dissociation constant]]s ''K''<sub>i</sub> or ''K''<sub>i</sub>', respectively.<ref name = "Bisswanger_2017" />{{rp|87}}
*Competitive inhibitors can bind to E, but not to ES. Competitive inhibition increases ''K''<sub>m</sub> (i.e., the inhibitor interferes with substrate binding), but does not affect ''V''<sub>max</sub> (the inhibitor does not hamper catalysis in ES because it cannot bind to ES).<ref name="Bisswanger_2017" />{{rp|102}}
*Uncompetitive inhibitors bind to ES. Uncompetitive inhibition decreases both ''K''<sub>m</sub> and ''V''<sub>max</sub>. The inhibitor affects substrate binding by increasing the enzyme's affinity for the substrate (decreasing ''K''<sub>m</sub>) as well as hampering catalysis (decreases ''V''<sub>max</sub>).<ref name="Bisswanger_2017" />{{rp|106}}
*Non-competitive inhibitors have identical affinities for E and ES (''K''<sub>i</sub> = ''K''<sub>i</sub>'). Non-competitive inhibition does not change ''K''<sub>m</sub> (i.e., it does not affect substrate binding) but decreases ''V''<sub>max</sub> (i.e., inhibitor binding hampers catalysis).<ref name="Bisswanger_2017" />{{rp|97}}
*Mixed-type inhibitors bind to both E and ES, but their affinities for these two forms of the enzyme are different (''K''<sub>i</sub> ≠ ''K''<sub>i</sub>'). Thus, mixed-type inhibitors affect substrate binding (increase or decrease ''K''<sub>m</sub>) and hamper catalysis in the ES complex (decrease ''V''<sub>max</sub>).<ref name="Marangoni_2003">{{cite book |title=Enzyme Kinetics: A Modern Approach |vauthors=Marangoni AG |date=2003 |publisher=Wiley-Interscience |isbn=978-0-471-15985-8 |location=Hoboken, N.J. |pages=61–69 |chapter=Reversible Enzyme Inhibition}}</ref>{{rp|63–64}}

When an enzyme has multiple substrates, inhibitors can show different types of inhibition depending on which substrate is considered. This results from the active site containing two different binding sites within the active site, one for each substrate. For example, an inhibitor might compete with substrate A for the first binding site, but be a non-competitive inhibitor with respect to substrate B in the second binding site.<ref>{{cite book | vauthors = Segel IH | title = Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems | publisher = Wiley–Interscience | edition = New | date = 1993 | isbn = 978-0-471-30309-1 }}</ref>

Traditionally reversible enzyme inhibitors have been classified as competitive, uncompetitive, or non-competitive, according to their effects on ''K''<sub>m</sub> and ''V''<sub>max</sub>.<ref name="Cleland_1963" /> These three types of inhibition result respectively from the inhibitor binding only to the enzyme E in the absence of substrate S, to the enzyme–substrate complex ES, or to both. The division of these classes arises from a problem in their derivation and results in the need to use two different binding constants for one binding event.<ref name = "Walsh_2021" /> It is further assumed that binding of the inhibitor to the enzyme results in 100% inhibition and fails to consider the possibility of partial inhibition.<ref name = "Walsh_2021">{{cite journal | vauthors = Walsh R, Martin E, Darvesh S | title = Limitations of conventional inhibitor classifications | journal = Integrative Biology | volume = 3 | issue = 12 | pages = 1197–1201 | date = December 2011 | pmid = 22038120 | doi = 10.1039/c1ib00053e }}</ref> The common form of the inhibitory term also obscures the relationship between the inhibitor binding to the enzyme and its relationship to any other binding term be it the Michaelis–Menten equation or a dose response curve associated with ligand receptor binding. To demonstrate the relationship the following rearrangement can be made:<ref name = "Walsh_2007" />{{rp|}}
:<math chem>\begin{align}\cfrac{V_\max}{1 + \cfrac\ce{[I]}{K_i}} &={V_\max}\left(\cfrac{K_i}{K_i+[\ce I]}\right)&&\text{multiply by } \cfrac{K_i}{K_i}=1\\
&={V_\max}\left(\cfrac{K_i+[\ce I]-[\ce I]}{K_i+[\ce I]}\right)&&\text{add }[\ce I]-[\ce I]=0\text{ to numerator}\\
&={V_\max}\left(1-\cfrac{[\ce I]}{K_i+[\ce I]}\right)&&\text{simplify }\cfrac{K_i+[\ce I]}{K_i+[\ce I]}=1\\
&=V_\max - V_\max \cfrac\ce{[I]}{K_i+[\ce I]}&&\text{multiply out by }V_\max \end{align}</math>

This rearrangement demonstrates that similar to the Michaelis–Menten equation, the maximal rate of reaction depends on the proportion of the enzyme population interacting with its substrate.

fraction of the enzyme population bound by substrate
:<math chem>\cfrac\ce{[S]}{[\ce S]+K_m}</math>

fraction of the enzyme population bound by inhibitor
:<math chem>\cfrac\ce{[I]}{[\ce I]+K_i}</math>

the effect of the inhibitor is a result of the percent of the enzyme population interacting with inhibitor. The only problem with this equation in its present form is that it assumes absolute inhibition of the enzyme with inhibitor binding, when in fact there can be a wide range of effects anywhere from 100% inhibition of substrate turn over to no inhibition. To account for this the equation can be easily modified to allow for different degrees of inhibition by including a delta ''V''<sub>max</sub> term.<ref name = "Walsh_2012">{{cite journal | vauthors = Walsh R | title = Alternative perspectives of enzyme kinetic modeling. | journal = Medicinal Chemistry and Drug Design. | publisher = InTech | date = May 2012 | issue = 16 | pages = 357–372 }}</ref>{{rp|361}}
:<math chem>V_\max - \Delta V_\max \cfrac\ce{[I]}{[\ce I]+K_i}</math>

or
:<math chem>V_{\max1} - (V_{\max1} - V_{\max2} ) \cfrac\ce{[I]}{[\ce I]+K_i}</math>

This term can then define the residual enzymatic activity present when the inhibitor is interacting with individual enzymes in the population. However the inclusion of this term has the added value of allowing for the possibility of activation if the secondary ''V''<sub>max</sub> term turns out to be higher than the initial term. To account for the possibly of activation as well the notation can then be rewritten replacing the inhibitor "I" with a modifier term (stimulator or inhibitor) denoted here as "X".<ref name = "Walsh_2007" />{{rp|eq 13}}
:<math chem>V_{\max1} - (V_{\max1} - V_{\max2} ) \cfrac\ce{[X]}{[\ce X]+K_x}</math>

While this terminology results in a simplified way of dealing with kinetic effects relating to the maximum velocity of the Michaelis–Menten equation, it highlights potential problems with the term used to describe effects relating to the ''K''<sub>m</sub>. The ''K''<sub>m</sub> relating to the affinity of the enzyme for the substrate should in most cases relate to potential changes in the binding site of the enzyme which would directly result from enzyme inhibitor interactions. As such a term similar to the delta ''V''<sub>max</sub> term proposed above to modulate ''V''<sub>max</sub> should be appropriate in most situations:<ref name = "Walsh_2007">{{cite journal | vauthors = Walsh R, Martin E, Darvesh S | title = A versatile equation to describe reversible enzyme inhibition and activation kinetics: modeling beta-galactosidase and butyrylcholinesterase | journal = Biochimica et Biophysica Acta (BBA) - General Subjects | volume = 1770 | issue = 5 | pages = 733–746 | date = May 2007 | pmid = 17307293 | doi = 10.1016/j.bbagen.2007.01.001 }}</ref>{{rp|eq 14}}
:<math chem>K_{m1} - (K_{m1} - K_{m2}) \cfrac\ce{[X]}{[\ce X]+K_x}</math>

===Dissociation constants===
[[File:Inhibition diagrams-1-.png|thumb|alt=2D plots of 1/[S] concentration (x-axis) and 1/V (y-axis) demonstrating that as inhibitor concentration is changed, competitive inhibitor lines intersect at a single point on the y-axis, non-competitive inhibitors intersect at the x-axis, and mixed inhibitors intersect a point that is on neither axis|Lineweaver–Burk diagrams of different types of reversible enzyme inhibitors. The arrow shows the effect of increasing concentrations of inhibitor.]]

An enzyme inhibitor is characterised by its [[dissociation constant]] ''K''<sub>i</sub>, the concentration at which the inhibitor half occupies the enzyme. In non-competitive inhibition the inhibitor can also bind to the enzyme-substrate complex, and the presence of bound substrate can change the affinity of the inhibitor for the enzyme, resulting in a second dissociation constant ''K''<sub>i</sub>'. Hence ''K''<sub>i</sub> and ''K''<sub>i</sub>' are the dissociation constants of the inhibitor for the enzyme and to the enzyme-substrate complex, respectively.<ref name = "Strelow_2012">{{cite book | vauthors = Strelow J, Dewe W, Iversen PW, Brooks PB, Radding JA, McGee J, Weidner J | date = October 2012 | chapter = Mechanism of Action Assays for Enzymes | veditors = Markossian S, Grossman A, Brimacombe K, etal | title = Assay Guidance Manual | chapter-url = https://www.ncbi.nlm.nih.gov/books/NBK92001/ | publisher = Eli Lilly & Company and the National Center for Advancing Translational Sciences | pmid = 22553872 | access-date = 9 April 2022 | archive-date = 15 June 2022 | archive-url = https://web.archive.org/web/20220615135104/http://www.ncbi.nlm.nih.gov/books/NBK92001/ | url-status = live }}</ref>{{rp|Glossary}} The enzyme-inhibitor constant ''K''<sub>i</sub> can be measured directly by various methods; one especially accurate method is [[Isothermal Titration Calorimetry|isothermal titration calorimetry]], in which the inhibitor is titrated into a solution of enzyme and the heat released or absorbed is measured.<ref>{{cite journal | vauthors = Holdgate GA | title = Making cool drugs hot: isothermal titration calorimetry as a tool to study binding energetics | journal = BioTechniques | volume = 31 | issue = 1 | pages = 164–6, 168, 170 passim | date = July 2001 | pmid = 11464510 }}</ref> However, the other dissociation constant ''K''<sub>i</sub>' is difficult to measure directly, since the enzyme-substrate complex is short-lived and undergoing a chemical reaction to form the product. Hence, ''K''<sub>i</sub>' is usually measured indirectly, by observing the [[enzyme activity]] under various substrate and inhibitor concentrations, and fitting the data via [[nonlinear regression]]<ref>{{cite journal | vauthors = Leatherbarrow RJ | title = Using linear and non-linear regression to fit biochemical data | journal = Trends in Biochemical Sciences | volume = 15 | issue = 12 | pages = 455–458 | date = December 1990 | pmid = 2077683 | doi = 10.1016/0968-0004(90)90295-M }}</ref> to a modified [[Enzyme kinetics|Michaelis–Menten equation]].<ref name = "Voet_2016">{{cite book | vauthors=Voet D, Voet JG, Pratt CW | chapter = Chapter 12: Enzyme Kinetics, Inhibition, and Control | pages = 361–401 |title=Fundamentals of Biochemistry: Life at the Molecular Level |date=2016 |publisher=Wiley |location=Hoboken, NJ |isbn=978-1-118-91840-1 |edition=Fifth}}</ref>
:<math>
V = \frac{V_{max}[S]}{\alpha K_{m} + \alpha^{\prime}[S]} = \frac{(1/\alpha^{\prime})V_{max}[S]}{(\alpha/\alpha^{\prime}) K_{m} + [S]}
</math>

where the modifying factors α and α' are defined by the inhibitor concentration and its two dissociation constants
:<math>
\alpha = 1 + \frac{[I]}{K_{i}}
</math>
:<math>
\alpha^{\prime} = 1 + \frac{[I]}{K_{i}^{\prime}}.
</math>

Thus, in the presence of the inhibitor, the enzyme's effective ''K''<sub>m</sub> and ''V''<sub>max</sub> become (α/α')''K''<sub>m</sub> and (1/α')''V''<sub>max</sub>, respectively. However, the modified Michaelis-Menten equation assumes that binding of the inhibitor to the enzyme has reached equilibrium, which may be a very slow process for inhibitors with sub-nanomolar dissociation constants. In these cases the inhibition becomes effectively irreversible, hence it is more practical to treat such tight-binding inhibitors as irreversible (see [[#Irreversible inhibitors|below]]).<!--{{cnspan|However, it can still be possible to estimate ''K''<sub>i</sub>' kinetically if ''K''<sub>i</sub> is measured independently.|date=July 2022}}-->

The effects of different types of reversible enzyme inhibitors on enzymatic activity can be visualised using graphical representations of the Michaelis–Menten equation, such as [[Lineweaver–Burk plot|Lineweaver–Burk]], [[Eadie-Hofstee diagram|Eadie-Hofstee]] or [[Hanes-Woolf plot]]s.<ref name = "Cornish-Bowden_2012" />{{rp|140–144}} An illustration is provided by the three Lineweaver–Burk plots depicted in the ''Lineweaver–Burk diagrams'' figure. In the top diagram the competitive inhibition lines intersect on the ''y''-axis, illustrating that such inhibitors do not affect ''V''<sub>max</sub>. In the bottom diagram the non-competitive inhibition lines intersect on the ''x''-axis, showing these inhibitors do not affect ''K''<sub>m</sub>. However, since it can be difficult to estimate ''K''<sub>i</sub> and ''K''<sub>i</sub>' accurately from such plots,<ref name = "Tseng_1990">{{cite journal | vauthors = Tseng SJ, Hsu JP | title = A comparison of the parameter estimating procedures for the Michaelis-Menten model | journal = Journal of Theoretical Biology | volume = 145 | issue = 4 | pages = 457–464 | date = August 1990 | pmid = 2246896 | doi = 10.1016/S0022-5193(05)80481-3 | bibcode = 1990JThBi.145..457T }}</ref> it is advisable to estimate these constants using more reliable nonlinear regression methods.<ref name = "Tseng_1990" />

===Special cases===
====Partially competitive====
The mechanism of partially competitive inhibition is similar to that of non-competitive, except that the EIS complex has catalytic activity, which may be lower or even higher (partially competitive activation) than that of the enzyme–substrate (ES) complex. This inhibition typically displays a lower ''V''<sub>max</sub>, but an unaffected ''K''<sub>m</sub> value.<ref name=Segel>{{cite book | vauthors = Segel IH | date = 1993 | title = Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems | publisher = Wiley-Interscience | edition = New | isbn = 978-0-471-30309-1 }}</ref>

====Substrate or product====
Substrate or product inhibition is where either an enzymes substrate or product also act as an inhibitor. This inhibition may follow the competitive, uncompetitive or mixed patterns. In substrate inhibition there is a progressive decrease in activity at high substrate concentrations, potentially from an enzyme having two competing substrate-binding sites. At low substrate, the high-affinity site is occupied and normal [[Enzyme kinetics|kinetics]] are followed. However, at higher concentrations, the second inhibitory site becomes occupied, inhibiting the enzyme.<ref>{{cite book | vauthors = Dixon M, Webb EC, Thorne CJ, Tipton KF | title = Enzymes | edition = 3rd | publisher = Longman | location = London | date = 1979 | page = 126| isbn = 978-0-470-20745-1 }}</ref> Product inhibition (either the enzyme's own product, or a product to an enzyme downstream in its metabolic pathway) is often a [[#Metabolic regulation|regulatory feature in metabolism]] and can be a form of [[negative feedback]].<ref name="Sauro_2017">{{cite journal | vauthors = Sauro HM | title = Control and regulation of pathways via negative feedback | journal = Journal of the Royal Society, Interface | volume = 14 | issue = 127 | pages = 1–13 | date = February 2017 | pmid = 28202588 | pmc = 5332569 | doi = 10.1098/rsif.2016.0848 }}</ref>

====Slow-tight====
Slow-tight inhibition occurs when the initial enzyme–inhibitor complex EI undergoes [[conformational isomerism]] (a change in shape) to a second more tightly held complex, EI*, but the overall inhibition process is reversible. This manifests itself as slowly increasing enzyme inhibition. Under these conditions, traditional Michaelis–Menten kinetics give a false value for ''K''<sub>i</sub>, which is time–dependent. The true value of ''K''<sub>i</sub> can be obtained through more complex analysis of the on (''k''<sub>on</sub>) and off (''k''<sub>off</sub>) rate constants for inhibitor association with kinetics similar to [[irreversible inhibition]].<ref name = "Cornish-Bowden_2012" />{{rp|168}}

====Multi-substrate analogues====
{{multiple image
| direction   = vertical
| image1      = TGDDF-GDDF.svg
| image2      = Ritonavir Schechter Berger Notation.svg
| image3      = Tipranavir structure 2.svg
| alt1        = TGDDF / GDDF MAIs where blue depicts the tetrahydrofolate cofactor analogue, black GAR or thioGAR and red, the connecting atoms.
| alt2        = Ritonavir is similar to the natural substrate.
| alt3        = Tipranavir is not similar to the natural substrate.
| caption1    = TGDDF/GDDF multi-substrate adduct inhibitor. Substrate analogue in black, cofactor analogue in blue, non-cleavable linker in red.
| caption2    = Peptide-based HIV-1 protease inhibitor [[ritonavir]] with substrate binding sites located in enzyme labelled as S2, S1, S1', and S2'.
| caption3    = Nonpeptidic HIV-1 protease inhibitor [[tipranavir]]
}}

Multi-substrate analogue inhibitors are high affinity selective inhibitors that can be prepared for enzymes that catalyse reactions with more than one substrate by capturing the binding energy of each of those substrate into one molecule.<ref>{{cite book | vauthors = Radzicka A, Wolfenden R | chapter = Transition state and multisubstrate analog inhibitors | title = Enzyme Kinetics and Mechanism Part D: Developments in Enzyme Dynamics | series = Methods in Enzymology | volume = 249 | pages = 284–312 | date = 1995 | pmid = 7791615 | doi = 10.1016/0076-6879(95)49039-6 | isbn = 9780121821500 }}</ref><ref>{{cite journal | vauthors = Schiffer CF, Burke JF, Besarab A, Lasker N, Simenhoff ML | title = Amylase/creatinine clearance fraction in patients on chronic hemodialysis | journal = Annals of Internal Medicine | volume = 86 | issue = 1 | pages = 65–66 | date = January 1977 | pmid = 319722 | doi = 10.7326/0003-4819-86-1-65 }}</ref> For example, in the [[Formylation|formyl transfer]] reactions of [[purine biosynthesis]], a potent Multi-substrate Adduct Inhibitor (MAI) to glycinamide ribonucleotide (GAR) [[Phosphoribosylglycinamide formyltransferase|TFase]] was prepared synthetically by linking analogues of the GAR substrate and the [[10-Formyltetrahydrofolate|N-10-formyl tetrahydrofolate]] cofactor together to produce thioglycinamide ribonucleotide dideazafolate (TGDDF),<ref>{{cite journal | vauthors = Inglese J, Blatchly RA, Benkovic SJ | title = A multisubstrate adduct inhibitor of a purine biosynthetic enzyme with a picomolar dissociation constant | journal = Journal of Medicinal Chemistry | volume = 32 | issue = 5 | pages = 937–940 | date = May 1989 | pmid = 2709379 | doi = 10.1021/jm00125a002 }}</ref> or enzymatically from the natural GAR substrate to yield GDDF.<ref>{{cite journal | title = Multisubstrate Adduct Inhibitors of Glycinamide Ribonucleotide Transformylase: Synthetic and Enzyme Generated. | vauthors = Inglese J, Benkovic SJ | journal = Tetrahedron|date = 1991 |volume = 47| issue = 14–15 | pages = 2351–2364| doi = 10.1016/S0040-4020(01)81773-7 }}</ref> Here the subnanomolar dissociation constant (KD) of TGDDF was greater than predicted presumably due to [[entropic]] advantages gained and/or positive interactions acquired through the atoms linking the components. MAIs have also been observed to be produced in cells by reactions of pro-drugs such as [[isoniazid]]<ref>{{cite journal | vauthors = Rozwarski DA, Grant GA, Barton DH, Jacobs WR, Sacchettini JC | title = Modification of the NADH of the isoniazid target (InhA) from Mycobacterium tuberculosis | journal = Science | volume = 279 | issue = 5347 | pages = 98–102 | date = January 1998 | pmid = 9417034 | doi = 10.1126/science.279.5347.98 | bibcode = 1998Sci...279...98R }}</ref> or enzyme inhibitor ligands (for example, [[PTC124]])<ref>{{cite journal | vauthors = Auld DS, Lovell S, Thorne N, Lea WA, Maloney DJ, Shen M, Rai G, Battaile KP, Thomas CJ, Simeonov A, Hanzlik RP, Inglese J | title = Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124 | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 107 | issue = 11 | pages = 4878–4883 | date = March 2010 | pmid = 20194791 | pmc = 2841876 | doi = 10.1073/pnas.0909141107 | doi-access = free | bibcode = 2010PNAS..107.4878A }}</ref> with cellular cofactors such as [[nicotinamide adenine dinucleotide]] (NADH) and [[adenosine triphosphate]] (ATP) respectively.<ref name="Le_Calvez_2009">{{cite journal | vauthors = Le Calvez PB, Scott CJ, Migaud ME | title = Multisubstrate adduct inhibitors: drug design and biological tools | journal = Journal of Enzyme Inhibition and Medicinal Chemistry | volume = 24 | issue = 6 | pages = 1291–318 | date = December 2009 | pmid = 19912064 | doi = 10.3109/14756360902843809 | s2cid = 21808708 }}</ref>

===Examples===
As enzymes have evolved to bind their substrates tightly, and most reversible inhibitors bind in the active site of enzymes, it is unsurprising that some of these inhibitors are strikingly similar in structure to the substrates of their targets. Inhibitors of [[dihydrofolate reductase]] (DHFR) are prominent examples.<ref name = "Avendano_2015">{{cite book | vauthors = Avendano C, Menendez JC | chapter = Chapter 2.5: Inhibitors of Dihydrofolate Reductase |title=Medicinal Chemistry of Anticancer Drugs |date=June 2015 |publisher=Elsevier |isbn=978-0-444-62667-7 |pages=54–58}}</ref> Other examples of these substrate mimics are the [[protease inhibitors]], a therapeutically effective class of [[antiretroviral drug]]s used to treat [[HIV/AIDS]].<ref>{{cite journal | vauthors = Hsu JT, Wang HC, Chen GW, Shih SR | title = Antiviral drug discovery targeting to viral proteases | journal = Current Pharmaceutical Design | volume = 12 | issue = 11 | pages = 1301–1314 | year = 2006 | pmid = 16611117 | doi = 10.2174/138161206776361110 }}</ref><ref name="Agbowuro_2018">{{cite journal | vauthors = Agbowuro AA, Huston WM, Gamble AB, Tyndall JD | title = Proteases and protease inhibitors in infectious diseases | journal = Medicinal Research Reviews | volume = 38 | issue = 4 | pages = 1295–1331 | date = July 2018 | pmid = 29149530 | doi = 10.1002/med.21475 | s2cid = 25269012 }}</ref> The structure of [[ritonavir]], a [[peptidomimetic]] (peptide mimic) protease inhibitor containing three [[peptide bond]]s, as shown in the [[#Competitive|"competitive inhibition"]] figure above. As this drug resembles the peptide that is the substrate of the HIV protease, it competes with the substrate in the enzyme's active site.<ref name="Qiu_2011">{{cite journal | vauthors = Qiu X, Liu ZP | title = Recent developments of peptidomimetic HIV-1 protease inhibitors | journal = Current Medicinal Chemistry | volume = 18 | issue = 29 | pages = 4513–37 | date = 2011 | pmid = 21864279 | doi = 10.2174/092986711797287566 }}</ref>

Enzyme inhibitors are often designed to mimic the [[transition state]] or intermediate of an enzyme-catalysed reaction.<ref name="Schramm_2018" /> This ensures that the inhibitor exploits the transition state stabilising effect of the enzyme, resulting in a better binding affinity (lower ''K''<sub>i</sub>) than substrate-based designs. An example of such a transition state inhibitor is the antiviral drug [[oseltamivir]]; this drug mimics the planar nature of the ring [[oxonium ion]] in the reaction of the viral enzyme [[neuraminidase]].<ref name="pmid10702632">{{cite journal | vauthors = Lew W, Chen X, Kim CU | title = Discovery and development of GS 4104 (oseltamivir): an orally active influenza neuraminidase inhibitor | journal = Current Medicinal Chemistry | volume = 7 | issue = 6 | pages = 663–672 | date = June 2000 | pmid = 10702632 | doi = 10.2174/0929867003374886 }}</ref>

However, not all inhibitors are based on the structures of substrates. For example, the structure of another HIV protease inhibitor [[tipranavir]] is not based on a peptide and has no obvious structural similarity to a protein substrate. These non-peptide inhibitors can be more stable than inhibitors containing peptide bonds, because they will not be substrates for [[peptidase]]s and are less likely to be degraded.<ref>{{cite journal | vauthors = Fischer PM | title = The design, synthesis and application of stereochemical and directional peptide isomers: a critical review | journal = Current Protein & Peptide Science | volume = 4 | issue = 5 | pages = 339–356 | date = October 2003 | pmid = 14529528 | doi = 10.2174/1389203033487054 }}</ref>

In drug design it is important to consider the concentrations of substrates to which the target enzymes are exposed. For example, some [[protein kinase]] inhibitors have chemical structures that are similar to ATP, one of the substrates of these enzymes.<ref name="Breen_2015">{{cite journal | vauthors = Breen ME, Soellner MB | title = Small molecule substrate phosphorylation site inhibitors of protein kinases: approaches and challenges | journal = ACS Chemical Biology | volume = 10 | issue = 1 | pages = 175–89 | date = January 2015 | pmid = 25494294 | pmc = 4301090 | doi = 10.1021/cb5008376 }}</ref> However, drugs that are simple competitive inhibitors will have to compete with the high concentrations of ATP in the cell. Protein kinases can also be inhibited by competition at the binding sites where the kinases interact with their substrate proteins, and most proteins are present inside cells at concentrations much lower than the concentration of ATP. As a consequence, if two protein kinase inhibitors both bind in the active site with similar affinity, but only one has to compete with ATP, then the competitive inhibitor at the protein-binding site will inhibit the enzyme more effectively.<ref>{{cite journal | vauthors = Bogoyevitch MA, Barr RK, Ketterman AJ | title = Peptide inhibitors of protein kinases-discovery, characterisation and use | journal = Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics | volume = 1754 | issue = 1–2 | pages = 79–99 | date = December 2005 | pmid = 16182621 | doi = 10.1016/j.bbapap.2005.07.025 }}</ref>

==Irreversible inhibitors==<!--This section is linked from [[Sarin]] and VX articles.-->

===Types===
{{multiple image
| header      = DFP reaction
| image1      = DIF reaction.png
| image2      = Inhibition schematic (irreversible).svg
| alt1        = 2D structural diagram depicting a serine amino acid residue from the active site of the enzyme forming a covalent bond with DFP by displacing the fluoride atom
| caption1    = Reaction of the irreversible inhibitor [[diisopropylfluorophosphate]] (DFP) with a [[serine protease]]
| caption2    = Irreversible inhibitors bind to the enzyme's binding site then undergo a chemical reaction to form a covalent enzyme-inhibitor complex (EI*). Binding site in blue, inhibitor in green.
}}

Irreversible inhibitors [[covalent]]ly bind to an enzyme, and this type of inhibition can therefore not be readily reversed.<ref name = "Patrick_2017">{{cite book |vauthors=Patrick GL |chapter=Enzymes as Drug Targets |title=An Introduction to Medicinal Chemistry |date=2017 |publisher=Oxford University Press |location=Oxford, United Kingdom |isbn=978-0-19-874969-1 |page=95 |edition=Sixth |chapter-url=https://books.google.com/books?id=bBqeDgAAQBAJ&pg=PA95 |access-date=3 June 2022 |archive-date=28 March 2023 |archive-url=https://web.archive.org/web/20230328010009/https://books.google.com/books?id=bBqeDgAAQBAJ&pg=PA95 |url-status=live }}</ref> Irreversible inhibitors often contain reactive functional groups such as [[nitrogen mustard]]s, [[aldehyde]]s, [[haloalkane]]s, [[alkene]]s, [[Michael acceptor]]s, [[Sulfonate|phenyl sulfonate]]s, or [[Methoxy arachidonyl fluorophosphonate|fluorophosphonate]]s.<ref name="Gehringer_2019">{{cite journal | vauthors = Gehringer M, Laufer SA | title = Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology | journal = Journal of Medicinal Chemistry | volume = 62 | issue = 12 | pages = 5673–5724 | date = June 2019 | pmid = 30565923 | doi = 10.1021/acs.jmedchem.8b01153 | s2cid = 56480231 }}</ref> These [[electrophilic]] groups react with amino acid side chains to form covalent [[adduct]]s.<ref name = "Patrick_2017" /> The residues modified are those with side chains containing [[nucleophile]]s such as [[hydroxyl]] or [[sulfhydryl]] groups; these include the amino acids [[serine]] (that reacts with [[Diisopropylfluorophosphate|DFP]], see the "DFP reaction" diagram), and also [[cysteine]], [[threonine]], or [[tyrosine]].<ref>{{cite book | vauthors = Lundblad RL |title = Chemical reagents for protein modification |publisher=CRC Press |isbn=978-0-8493-1983-9 | date = 2004 |edition=3rd }}</ref>

Irreversible inhibition is different from irreversible enzyme inactivation.<ref>{{cite journal | vauthors = Polakovič M, Vrabel P, Báleš V | title = Approaches for improved identification of mechanisms of enzyme inactivation. | journal = Progress in Biotechnology | date = January 1998 | volume = 15 | pages = 77–82 | publisher = Elsevier | isbn = 978-0-444-82970-2 | doi = 10.1016/S0921-0423(98)80013-0 | quote = Enzyme inactivation is generally explained as a chemical process involving several phenomena like aggregation, dissociation into subunits, or denaturation (conformational changes), which occur simultaneously during the inactivation of a specific enzyme. }}</ref> Irreversible inhibitors are generally specific for one class of enzyme and do not inactivate all proteins; they do not function by destroying [[protein structure]] but by specifically altering the active site of their target. For example, extremes of pH or temperature usually cause [[Denaturation (biochemistry)|denaturation]] of all protein structure, but this is a non-specific effect. Similarly, some non-specific chemical treatments destroy protein structure: for example, heating in concentrated [[hydrochloric acid]] will hydrolyse the [[peptide bond]]s holding proteins together, releasing free amino acids.<ref>{{cite book | vauthors = Price N, Hames B, Rickwood D |title=Proteins LabFax |publisher=BIOS Scientific Publishers |isbn=978-0-12-564710-6|year=1996 }}</ref>

Irreversible inhibitors display time-dependent inhibition and their potency therefore cannot be characterised by an IC<sub>50</sub> value. This is because the amount of active enzyme at a given concentration of irreversible inhibitor will be different depending on how long the inhibitor is pre-incubated with the enzyme. Instead, ''k''<sub>obs</sub>/[''I''] values are used,<ref>{{cite journal | vauthors = Adam GC, Cravatt BF, Sorensen EJ | title = Profiling the specific reactivity of the proteome with non-directed activity-based probes | journal = Chemistry & Biology | volume = 8 | issue = 1 | pages = 81–95 | date = January 2001 | pmid = 11182321 | doi = 10.1016/S1074-5521(00)90060-7 | doi-access = free }}</ref> where ''k''<sub>obs</sub> is the observed pseudo-first order rate of inactivation (obtained by plotting the log of % activity versus time) and [''I''] is the concentration of inhibitor. The ''k''<sub>obs</sub>/[''I''] parameter is valid as long as the inhibitor does not saturate binding with the enzyme (in which case ''k''<sub>obs</sub> = ''k''<sub>inact</sub>) where ''k''<sub>inact</sub> is the rate of inactivation.

===Measuring===
{{multiple image
| image1            = Inhibition mechanism (irreversible).svg
| header            = Irreversible inhibition mechanism
| alt1              = Depiction of the reversible chemical equilibria between enzyme + substrate, enzyme/substrate complex, and enzyme + product, and two competing equilibria. The first is between enzyme + inhibitor, enzyme/inhibitor non-covalent complex, followed by irreversible formation of the covalent complex. The second is between enzyme/substrate complex + inhibitor, noncovalent enzyme/substrate, followed by irreversible formation of the covalent complex
| caption1          = Kinetic mechanism for irreversible inhibition. Substrate binding in blue, catalysis in red, inhibitor binding in green, inactivation reaction in dark green.

}}
Irreversible inhibitors first form a reversible non-covalent complex with the enzyme (EI or ESI). Subsequently, a chemical reaction occurs between the enzyme and inhibitor to produce the covalently modified "dead-end complex" EI* (an irreversible covalent complex). The rate at which EI* is formed is called the inactivation rate or ''k''<sub>inact</sub>.<ref name="Tuley_2018" /> Since formation of EI may compete with ES, binding of irreversible inhibitors can be prevented by competition either with substrate or with a second, reversible inhibitor. This protection effect is good evidence of a specific reaction of the irreversible inhibitor with the active site.

The binding and inactivation steps of this reaction are investigated by incubating the enzyme with inhibitor and assaying the amount of activity remaining over time. The activity will be decreased in a time-dependent manner, usually following [[exponential decay]]. Fitting these data to a [[rate equation]] gives the rate of inactivation at this concentration of inhibitor. This is done at several different concentrations of inhibitor. If a reversible EI complex is involved the inactivation rate will be saturable and fitting this curve will give ''k''<sub>inact</sub> and ''K''<sub>i</sub>.<ref>{{cite journal | vauthors = Maurer T, Fung HL | title = Comparison of methods for analyzing kinetic data from mechanism-based enzyme inactivation: application to nitric oxide synthase | journal = AAPS PharmSci | volume = 2 | issue = 1 | pages = 68–77 | year = 2000 | pmid = 11741224 | pmc = 2751003 | doi = 10.1208/ps020108 }}</ref>

Another method that is widely used in these analyses is [[mass spectrometry]]. Here, accurate measurement of the mass of the unmodified native enzyme and the inactivated enzyme gives the increase in mass caused by reaction with the inhibitor and shows the stoichiometry of the reaction.<ref name="pmid10398927">{{cite journal | vauthors = Loo JA, DeJohn DE, Du P, Stevenson TI, Ogorzalek Loo RR | title = Application of mass spectrometry for target identification and characterization | journal = Medicinal Research Reviews | volume = 19 | issue = 4 | pages = 307–319 | date = July 1999 | pmid = 10398927 | doi = 10.1002/(SICI)1098-1128(199907)19:4<307::AID-MED4>3.0.CO;2-2 | s2cid = 11766917 }}</ref> This is usually done using a [[MALDI-TOF]] mass spectrometer.<ref>{{cite book |vauthors=Purich DL |title=Enzyme Kinetics: Catalysis and Control: A Reference of Theory and Best-Practice Methods |date=2010 |publisher=Elsevier Academic |location=San Diego, Calif. |isbn=978-0-12-380925-4 |page=542 |chapter=Irreversible Enzyme Inhibition by Affinity Labelling Agents |chapter-url=https://books.google.com/books?id=SkSQNNACcrYC&dq=irreversible+enzyme+inhibitor+MALDI-TOF&pg=PA542 |access-date=20 July 2022 |archive-date=20 July 2022 |archive-url=https://web.archive.org/web/20220720192921/https://books.google.com/books?id=SkSQNNACcrYC&dq=irreversible+enzyme+inhibitor+MALDI-TOF&pg=PA542 |url-status=live }}</ref> In a complementary technique, [[peptide mass fingerprinting]] involves digestion of the native and modified protein with a [[protease]] such as [[trypsin]]. This will produce a set of [[peptide]]s that can be analysed using a mass spectrometer. The peptide that changes in mass after reaction with the inhibitor will be the one that contains the site of modification.<ref name="Sibille_2012">{{cite journal | vauthors = Sibille E, Bana E, Chaouni W, Diederich M, Bagrel D, Chaimbault P | title = Development of a matrix-assisted laser desorption/ionization-mass spectrometry screening test to evidence reversible and irreversible inhibitors of CDC25 phosphatases | journal = Analytical Biochemistry | volume = 430 | issue = 1 | pages = 83–91 | date = November 2012 | pmid = 22902804 | doi = 10.1016/j.ab.2012.08.006 }}</ref>

===Slow binding===
[[File:DFMO inhibitor mechanism.svg|thumb|alt=2D chemical structure diagram depicting a lysine residue from the enzyme first reacting with DFMO, elimination of fluoride and carbon dioxide, followed by cysteine attacking the covalent lysine-DFMO adduct freeing the lysine residue to form an irreversible cysteine adduct|Chemical mechanism for irreversible inhibition of ornithine decarboxylase by DFMO. Pyridoxal 5'-phosphate (Py) and enzyme (E) are not shown. Adapted from Poulin ''et al'', 1992.<ref name=Poulin>{{cite journal | vauthors = Poulin R, Lu L, Ackermann B, Bey P, Pegg AE | title = Mechanism of the irreversible inactivation of mouse ornithine decarboxylase by alpha-difluoromethylornithine. Characterization of sequences at the inhibitor and coenzyme binding sites | journal = The Journal of Biological Chemistry | volume = 267 | issue = 1 | pages = 150–158 | date = January 1992 | pmid = 1730582 | doi = 10.1016/S0021-9258(18)48472-4 | doi-access = free }}</ref>]]

Not all irreversible inhibitors form covalent adducts with their enzyme targets. Some reversible inhibitors bind so tightly to their target enzyme that they are essentially irreversible. These tight-binding inhibitors may show kinetics similar to covalent irreversible inhibitors. In these cases some of these inhibitors rapidly bind to the enzyme in a low-affinity EI complex and this then undergoes a slower rearrangement to a very tightly bound EI* complex (see the "irreversible inhibition mechanism" diagram). This kinetic behaviour is called slow-binding.<ref>{{cite book | vauthors = Szedlacsek SE, Duggleby RG | title = Kinetics of slow and tight-binding inhibitors | series = Methods in Enzymology | volume = 249 | pages = 144–80 | year = 1995 | pmid = 7791610 | doi = 10.1016/0076-6879(95)49034-5 | isbn = 978-0-12-182150-0 | chapter = &#91;6&#93; Kinetics of slow and tight-binding inhibitors }}</ref> This slow rearrangement after binding often involves a [[conformational change]] as the enzyme "clamps down" around the inhibitor molecule. Examples of slow-binding inhibitors include some important drugs, such [[methotrexate]],<ref>{{cite journal | vauthors = Stone SR, Morrison JF | title = Mechanism of inhibition of dihydrofolate reductases from bacterial and vertebrate sources by various classes of folate analogs | journal = Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology | volume = 869 | issue = 3 | pages = 275–285 | date = February 1986 | pmid = 3511964 | doi = 10.1016/0167-4838(86)90067-1 }}</ref> [[allopurinol]],<ref>{{cite journal | vauthors = Pick FM, McGartoll MA, Bray RC | title = Reaction of formaldehyde and of methanol with xanthine oxidase | journal = European Journal of Biochemistry | volume = 18 | issue = 1 | pages = 65–72 | date = January 1971 | pmid = 4322209 | doi = 10.1111/j.1432-1033.1971.tb01215.x | doi-access = free }}</ref> and the activated form of [[acyclovir]].<ref>{{cite journal | vauthors = Reardon JE | title = Herpes simplex virus type 1 and human DNA polymerase interactions with 2'-deoxyguanosine 5'-triphosphate analogs. Kinetics of incorporation into DNA and induction of inhibition | journal = The Journal of Biological Chemistry | volume = 264 | issue = 32 | pages = 19039–19044 | date = November 1989 | pmid = 2553730 | doi = 10.1016/S0021-9258(19)47263-3 | doi-access = free }}</ref>

===Some examples===
[[File:PDB 1GXF.png|thumb|[[Trypanothione|Trypanothione reductase]] with the lower molecule of an inhibitor bound irreversibly and the upper one reversibly. Created from Bond ''et al'', 2004.<ref name="pmid15102853">{{PDB|1GXF}}; {{cite journal | vauthors = Saravanamuthu A, Vickers TJ, Bond CS, Peterson MR, Hunter WN, Fairlamb AH | title = Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design | journal = The Journal of Biological Chemistry | volume = 279 | issue = 28 | pages = 29493–500 | date = July 2004 | pmid = 15102853 | pmc = 3491871 | doi = 10.1074/jbc.M403187200 | doi-access = free }}</ref> ({{Pdb|1GXF}})|alt=3D cartoon diagram of the trypanothione reductase protein bound to two molecules of inhibitors depicted as a stick diagrams.]]

[[Diisopropylfluorophosphate]] (DFP) is an example of an irreversible [[Protease inhibitor (pharmacology)|protease inhibitor]] (see the "DFP reaction" diagram). The enzyme hydrolyses the phosphorus–fluorine bond, but the phosphate residue remains bound to the serine in the [[Catalytic triad|active site]], deactivating it.<ref>{{cite book | vauthors = Cohen JA, Oosterbaan RA, Berends F | title = Enzyme Structure |volume=11 |pages=686–702 |year=1967 |doi=10.1016/S0076-6879(67)11085-9 |chapter= [81] Organophosphorus compounds|series=Methods in Enzymology|isbn=978-0-12-181860-9 | archive-url = https://web.archive.org/web/20180228070651/http://oai.dtic.mil/oai/oai | archive-date = 28 February 2018 | chapter-url = https://oai.dtic.mil/oai/oai|url-status=dead }}</ref> Similarly, DFP also reacts with the active site of [[acetylcholine esterase]] in the [[synapses]] of neurons, and consequently is a potent neurotoxin, with a lethal dose of less than 100{{spaces}}mg.<ref>{{cite book | vauthors = Brenner GM |title=Pharmacology | location = Philadelphia, PA |publisher=W.B. Saunders |isbn=978-0-7216-7757-6 | year = 2000 |edition=1st }}</ref>

[[Suicide inhibition]] is an unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive form in its active site.<ref name="Walsh_1984">{{cite journal | vauthors = Walsh CT | title = Suicide substrates, mechanism-based enzyme inactivators: recent developments | journal = Annual Review of Biochemistry | volume = 53 | issue = | pages = 493–535 | date = 1984 | pmid = 6433782 | doi = 10.1146/annurev.bi.53.070184.002425 }}</ref> An example is the inhibitor of [[polyamine]] biosynthesis, [[Eflornithine|α-difluoromethylornithine]] (DFMO), which is an analogue of the amino acid [[ornithine]], and is used to treat [[African trypanosomiasis]] (sleeping sickness). [[Ornithine decarboxylase]] can catalyse the decarboxylation of DFMO instead of ornithine (see the "DFMO inhibitor mechanism" diagram). However, this decarboxylation reaction is followed by the elimination of a fluorine atom, which converts this catalytic intermediate into a conjugated [[imine]], a highly electrophilic species. This reactive form of DFMO then reacts with either a cysteine or lysine residue in the active site to irreversibly inactivate the enzyme.<ref name=Poulin/>

Since irreversible inhibition often involves the initial formation of a non-covalent enzyme inhibitor (EI) complex,<ref name="Tuley_2018" /> it is sometimes possible for an inhibitor to bind to an enzyme in more than one way. For example, in the figure showing [[Trypanothione|trypanothione reductase]] from the human protozoan parasite ''[[Trypanosoma cruzi]]'', two molecules of an inhibitor called ''quinacrine mustard'' are bound in its active site. The top molecule is bound reversibly, but the lower one is bound covalently as it has reacted with an amino acid residue through its [[nitrogen mustard]] group.<ref>{{cite journal | vauthors = Saravanamuthu A, Vickers TJ, Bond CS, Peterson MR, Hunter WN, Fairlamb AH | title = Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design | journal = The Journal of Biological Chemistry | volume = 279 | issue = 28 | pages = 29493–29500 | date = July 2004 | pmid = 15102853 | pmc = 3491871 | doi = 10.1074/jbc.M403187200 | doi-access = free }}</ref>

==Applications==
Enzyme inhibitors are found in nature<ref>{{cite book | vauthors = Pereira DM, Andrade C, Valentão P, Andrade PB | chapter = Natural products as enzyme inhibitors. | title = Natural Products Targeting Clinically Relevant Enzymes | edition = First | date = October 2017 | publisher = Wiley-VCH Verlag GmbH & Co. KGaA | isbn = 978-3-527-34205-1 | chapter-url = https://application.wiley-vch.de/books/sample/3527342052_c01.pdf | access-date = 3 April 2022 | archive-date = 5 December 2022 | archive-url = https://web.archive.org/web/20221205143202/https://application.wiley-vch.de/books/sample/3527342052_c01.pdf | url-status = live }}</ref> and also produced artificially in the laboratory.<ref name="Hiratake_2005">{{cite journal | vauthors = Hiratake J | title = Enzyme inhibitors as chemical tools to study enzyme catalysis: rational design, synthesis, and applications | journal = Chemical Record | location = New York, N.Y. | volume = 5 | issue = 4 | pages = 209–28 | date = 2005 | pmid = 16041744 | doi = 10.1002/tcr.20045 }}</ref> Naturally occurring enzyme inhibitors regulate many [[metabolic]] processes and are essential for life.<ref name = "Plaxton_2004">{{cite book |vauthors=Plaxton WC |chapter=Principles of Metabolic Control |veditors=Storey KB |title=Functional Metabolism: Regulation and Adaptation |date=2004 |publisher=John Wiley & Sons |location=Hoboken, N.J. |isbn=978-0-471-67557-0 |pages=1–24 (12) |chapter-url=https://books.google.com/books?id=d1nu4vcml8sC&q=inhibitor&pg=PA11 |access-date=14 April 2022 |archive-date=28 March 2023 |archive-url=https://web.archive.org/web/20230328010003/https://books.google.com/books?id=d1nu4vcml8sC&q=inhibitor&pg=PA11 |url-status=live }}</ref><ref name = "Copeland_2013" /> In addition, naturally produced [[poison]]s are often enzyme inhibitors that have evolved for use as toxic agents against predators, prey, and competing organisms.<ref name="Haefner_2003">{{cite journal | vauthors = Haefner B | title = Drugs from the deep: marine natural products as drug candidates | journal = Drug Discovery Today | volume = 8 | issue = 12 | pages = 536–44 | date = June 2003 | pmid = 12821301 | doi = 10.1016/s1359-6446(03)02713-2 }}</ref> These natural toxins include some of the most poisonous substances known.<ref>{{cite book |vauthors=Page C, Pitchford S |chapter=Venoms, toxins, poisons and herbs |title=Dale's Pharmacology Condensed E-Book |date=2021 |publisher=Elsevier Health Sciences |location=Philadelphia, Pa. |isbn=978-0-7020-7819-4 |pages=153–155 |edition=Third |chapter-url=https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780702078187000352 |access-date=3 June 2022 |archive-date=25 April 2013 |archive-url=https://web.archive.org/web/20130425174959/https://www.clinicalkey.com/#!/content/book/3-s2.0-B9780702078187000352 |url-status=live }}</ref> Artificial inhibitors are often used as drugs, but can also be [[insecticide]]s such as [[malathion]], [[herbicide]]s such as [[glyphosate]],<ref>{{cite book |vauthors=Stenersen J |chapter=Chapter 5: Specific Enzyme Inhibitors |chapter-url=https://books.google.com/books?id=WjReuSXxl4YC&pg=PA82 |title=Chemical Pesticides Mode of Action and Toxicology |date=2004 |publisher=CRC Press |location=Boca Raton |isbn=978-0-203-64683-0 |pages=73–114 |access-date=14 April 2022 |archive-date=28 March 2023 |archive-url=https://web.archive.org/web/20230328005931/https://books.google.com/books?id=WjReuSXxl4YC&pg=PA82 |url-status=live }}</ref> or [[disinfectant]]s such as [[triclosan]]. Other artificial enzyme inhibitors block [[acetylcholinesterase]], an enzyme which breaks down [[acetylcholine]], and are used as [[nerve agent]]s in [[chemical warfare]].<ref>{{cite book | vauthors = Greathouse B, Zahra F, Brady MF | chapter = Acetylcholinesterase Inhibitors Toxicity | chapter-url = https://www.ncbi.nlm.nih.gov/books/NBK535428/ | date = September 2021 | title = StatPearls [Internet] | location = Treasure Island (FL) | publisher = StatPearls Publishing | pmid = 30571049 | access-date = 14 April 2022 | archive-date = 17 January 2023 | archive-url = https://web.archive.org/web/20230117033429/https://www.ncbi.nlm.nih.gov/books/NBK535428/ | url-status = live }}</ref>

===Metabolic regulation===
Enzyme inhibition is a common feature of [[metabolic pathway]] control in cells.<ref name = "Plaxton_2004" /> [[Metabolic flux]] through a pathway is often regulated by a pathway's [[metabolites]] acting as inhibitors and enhancers for the enzymes in that same pathway. The [[Glycolysis|glycolytic pathway]] is a classic example.<ref name="Orencio-Trejo_2010">{{cite book | vauthors = Orencio-Trejo M, Utrilla J, Fernández-Sandoval MT, Huerta-Beristain G, Gosset G, Martinez A | chapter = Engineering the Escherichia coli Fermentative Metabolism | veditors = Cordes M, Wittmann C, Krull R |title=Biosystems Engineering II: Linking Cellular Networks and Bioprocesses |date=2010 |publisher=Springer Science & Business Media |location=Berlin |isbn=978-3-642-13865-2 |pages=77–78 |chapter-url=https://books.google.com/books?id=TMhreJSpuU8C&pg=PA77 }}</ref> This [[Catabolism|catabolic]] pathway consumes [[glucose]] and produces [[Adenosine triphosphate|ATP]], [[Nicotinamide adenine dinucleotide|NADH]] and [[pyruvate]]. A key step for the regulation of glycolysis is an early reaction in the pathway catalysed by [[Phosphofructokinase|phosphofructokinase{{nbh}}1]] (PFK1). When ATP levels rise, ATP binds an allosteric site in PFK1 to decrease the rate of the enzyme reaction; glycolysis is inhibited and ATP production falls. This [[Feedback|negative feedback]] control helps maintain a steady concentration of ATP in the cell. However, metabolic pathways are not just regulated through inhibition since enzyme activation is equally important. With respect to PFK1, [[fructose 2,6-bisphosphate]] and [[Adenosine diphosphate|ADP]] are examples of metabolites that are allosteric activators.<ref>{{cite journal | vauthors = Okar DA, Lange AJ | title = Fructose-2,6-bisphosphate and control of carbohydrate metabolism in eukaryotes | journal = BioFactors | volume = 10 | issue = 1 | pages = 1–14 | year = 1999 | pmid = 10475585 | doi = 10.1002/biof.5520100101 | s2cid = 24586866 }}</ref>

Physiological enzyme inhibition can also be produced by specific protein inhibitors. This mechanism occurs in the [[pancreas]], which synthesises many digestive precursor enzymes known as [[zymogen]]s. Many of these are activated by the [[trypsin]] protease, so it is important to inhibit the activity of trypsin in the pancreas to prevent the organ from digesting itself. One way in which the activity of trypsin is controlled is the production of a specific and potent [[trypsin inhibitor]] protein in the pancreas. This inhibitor binds tightly to trypsin, preventing the trypsin activity that would otherwise be detrimental to the organ.<ref>{{cite book | vauthors = Price NC, Stevens L |title=Fundamentals of enzymology : the cell and molecular biology of catalytic proteins |publisher=Oxford University Press |isbn=978-0-19-850229-6 |edition=3rd | year = 1999 }}</ref> Although the trypsin inhibitor is a protein, it avoids being hydrolysed as a substrate by the protease by excluding water from trypsin's active site and destabilising the transition state.<ref>{{cite journal | vauthors = Smyth TP | title = Substrate variants versus transition state analogues as noncovalent reversible enzyme inhibitors | journal = Bioorganic & Medicinal Chemistry | volume = 12 | issue = 15 | pages = 4081–4088 | date = August 2004 | pmid = 15246086 | doi = 10.1016/j.bmc.2004.05.041 | url = https://zenodo.org/record/998967 | access-date = 14 April 2022 | archive-date = 28 March 2023 | archive-url = https://web.archive.org/web/20230328005934/https://zenodo.org/record/998967 | url-status = live }}</ref> Other examples of physiological enzyme inhibitor proteins include the [[barstar]] inhibitor of the bacterial ribonuclease [[barnase]].<ref>{{cite journal | vauthors = Hartley RW | title = Barnase and barstar: two small proteins to fold and fit together | journal = Trends in Biochemical Sciences | volume = 14 | issue = 11 | pages = 450–454 | date = November 1989 | pmid = 2696173 | doi = 10.1016/0968-0004(89)90104-7 }}</ref>

===Natural poisons===
[[File:3 types of lentil.jpg|thumb|alt=photo of three piles of legume seeds coloured brown, pea green, and brown/orange|To discourage [[seed predation]], [[legume]]s contain [[trypsin inhibitor]]s that interfere with digestion.]]

Animals and plants have evolved to synthesise a vast array of poisonous products including [[secondary metabolite]]s,<ref>{{cite book | vauthors = Maheshwari VL |title=Natural Products as Enzyme Inhibitors |date=2022 |publisher=Springer |location=Singapore |isbn=978-981-19-0932-0}}</ref> peptides and proteins<ref>{{cite book | vauthors = Birk Y |title=Plant Protease Inhibitors: Significance in Nutrition, Plant Protection, Cancer Prevention and Genetic Engineering |date=2003 |publisher=Springer |location=Berlin |isbn=978-3-540-00118-8}}</ref> that can act as inhibitors. Natural toxins are usually [[Small molecule|small organic molecules]] and are so diverse that there are probably natural inhibitors for most metabolic processes.<ref>{{cite journal | vauthors = Tan G, Gyllenhaal C, Soejarto DD | title = Biodiversity as a source of anticancer drugs | journal = Current Drug Targets | volume = 7 | issue = 3 | pages = 265–277 | date = March 2006 | pmid = 16515527 | doi = 10.2174/138945006776054942 }}</ref> The metabolic processes targeted by natural poisons encompass more than enzymes in metabolic pathways and can also include the inhibition of receptor, channel and structural protein functions in a cell. For example, [[paclitaxel]] (taxol), an organic molecule found in the [[Taxus|Pacific yew tree]], binds tightly to [[tubulin]] [[Protein dimer|dimers]] and inhibits their assembly into [[microtubule]]s in the [[cytoskeleton]].<ref>{{cite journal | vauthors = Abal M, Andreu JM, Barasoain I | title = Taxanes: microtubule and centrosome targets, and cell cycle dependent mechanisms of action | journal = Current Cancer Drug Targets | volume = 3 | issue = 3 | pages = 193–203 | date = June 2003 | pmid = 12769688 | doi = 10.2174/1568009033481967 }}</ref>

Many natural poisons act as [[neurotoxin]]s that can cause [[paralysis]] leading to death and function for defence against predators or in hunting and capturing prey. Some of these natural inhibitors,<ref name = "Hostettmann_2006">{{cite journal | vauthors = Hostettmann K, Borloz A, Urbain A, Marston A | title = Natural Product Inhibitors of Acetylcholinesterase |journal=Current Organic Chemistry |volume=10 |issue=8 |pages=825–847 |year=2006 |doi=10.2174/138527206776894410 }}</ref> despite their toxic attributes, are valuable for therapeutic uses at lower doses.<ref name="Knight_2018">{{cite journal | vauthors = Knight R, Khondoker M, Magill N, Stewart R, Landau S | title = A Systematic Review and Meta-Analysis of the Effectiveness of Acetylcholinesterase Inhibitors and Memantine in Treating the Cognitive Symptoms of Dementia | journal = Dementia and Geriatric Cognitive Disorders | volume = 45 | issue = 3–4 | pages = 131–151 | date = 2018 | pmid = 29734182 | doi = 10.1159/000486546 | s2cid = 13701908 | doi-access = free }}</ref> An example of a neurotoxin are the [[glycoalkaloid]]s, from the plant species in the family [[Solanaceae]] (includes [[potato]], [[tomato]] and [[eggplant]]), that are [[acetylcholinesterase inhibitor]]s. Inhibition of this enzyme causes an uncontrolled increase in the acetylcholine neurotransmitter, muscular paralysis and then death. [[Neurotoxicity]] can also result from the inhibition of receptors; for example, [[atropine]] from deadly nightshade (''[[Atropa belladonna]]'') that functions as a [[competitive antagonist]] of the [[Acetylcholine receptor|muscarinic acetylcholine receptors]].<ref>{{cite journal | vauthors = DeFrates LJ, Hoehns JD, Sakornbut EL, Glascock DG, Tew AR | title = Antimuscarinic intoxication resulting from the ingestion of moonflower seeds | journal = The Annals of Pharmacotherapy | volume = 39 | issue = 1 | pages = 173–176 | date = January 2005 | pmid = 15572604 | doi = 10.1345/aph.1D536 | s2cid = 36465515 }}</ref>

Although many natural toxins are secondary metabolites, these poisons also include peptides and proteins. An example of a toxic peptide is [[alpha-amanitin]], which is found in relatives of the [[death cap]] mushroom. This is a potent enzyme inhibitor, in this case preventing the [[RNA polymerase&nbsp;II]] enzyme from transcribing DNA.<ref>{{cite journal | vauthors = Vetter J | title = Toxins of Amanita phalloides | journal = Toxicon | volume = 36 | issue = 1 | pages = 13–24 | date = January 1998 | pmid = 9604278 | doi = 10.1016/S0041-0101(97)00074-3 | bibcode = 1998Txcn...36...13V }}</ref> The algal toxin [[microcystin]] is also a peptide and is an inhibitor of [[Phosphatase|protein phosphatases]].<ref>{{cite journal | vauthors = Holmes CF, Maynes JT, Perreault KR, Dawson JF, James MN | title = Molecular enzymology underlying regulation of protein phosphatase-1 by natural toxins | journal = Current Medicinal Chemistry | volume = 9 | issue = 22 | pages = 1981–1989 | date = November 2002 | pmid = 12369866 | doi = 10.2174/0929867023368827 }}</ref> This toxin can contaminate water supplies after [[algal bloom]]s and is a known carcinogen that can also cause acute liver haemorrhage and death at higher doses.<ref>{{cite journal | vauthors = Bischoff K | title = The toxicology of microcystin-LR: occurrence, toxicokinetics, toxicodynamics, diagnosis and treatment | journal = Veterinary and Human Toxicology | volume = 43 | issue = 5 | pages = 294–297 | date = October 2001 | pmid = 11577938 }}</ref>

Proteins can also be natural poisons or [[antinutrient]]s, such as the [[trypsin inhibitor]]s (discussed in the "metabolic regulation" section above) that are found in some [[Pulse (legume)|legume]]s.<ref>{{cite book | vauthors = Savage GP, Morrison SC | chapter = Trypsin inhibitors. | veditors = Caballero B | title = Encyclopedia of Food Sciences and Nutrition | year = 2003 | edition = Second | pages = 5878–5884 | doi = 10.1016/B0-12-227055-X/00934-2 | isbn = 978-0-12-227055-0 | publisher = Academic Press }}</ref> A less common class of toxins are toxic enzymes: these act as irreversible inhibitors of their target enzymes and work by chemically modifying their substrate enzymes. An example is [[ricin]], an extremely potent protein toxin found in [[Castor oil plant|castor oil beans]].<ref name="Polito_2019">{{cite journal | vauthors = Polito L, Bortolotti M, Battelli MG, Calafato G, Bolognesi A | title = Ricin: An Ancient Story for a Timeless Plant Toxin | journal = Toxins | volume = 11 | issue = 6 | date = June 2019 | page = 324 | pmid = 31174319 | pmc = 6628454 | doi = 10.3390/toxins11060324 | doi-access = free }}</ref> This enzyme is a [[glycosidase]] that inactivates ribosomes.<ref name="Sowa-Rogozińska_2019">{{cite journal | vauthors = Sowa-Rogozińska N, Sominka H, Nowakowska-Gołacka J, Sandvig K, Słomińska-Wojewódzka M | title = Intracellular Transport and Cytotoxicity of the Protein Toxin Ricin | journal = Toxins | volume = 11 | issue = 6 | date = June 2019 | page = 350 | pmid = 31216687 | pmc = 6628406 | doi = 10.3390/toxins11060350 | doi-access = free }}</ref> Since ricin is a catalytic irreversible inhibitor, this allows just a single molecule of ricin to kill a cell.<ref>{{cite journal | vauthors = Hartley MR, Lord JM | title = Cytotoxic ribosome-inactivating lectins from plants | journal = Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics | volume = 1701 | issue = 1–2 | pages = 1–14 | date = September 2004 | pmid = 15450171 | doi = 10.1016/j.bbapap.2004.06.004 }}</ref>

===Drugs===
[[File:Methotrexate vs folate.svg|thumb|alt=2D chemical structural diagrams comparing folic acid and methotrexate|The coenzyme folic acid (top) compared to the anti-cancer drug methotrexate (bottom)]]
[[File:Sildenafil.svg|thumb|alt=2D structural diagram of sildenafil|The structure of [[sildenafil]] (Viagra)]]

The most common uses for enzyme inhibitors are as drugs to treat disease. Many of these inhibitors target a human enzyme and aim to correct a pathological condition. For instance, [[aspirin]] is a widely used drug that acts as a [[suicide inhibitor]] of the [[cyclooxygenase]] enzyme.<ref name="Finkel_2009" /> This inhibition in turn suppresses the production of proinflammatory [[prostaglandin]]s and thus aspirin may be used to reduce pain, fever, and inflammation.<ref name="Finkel_2009">{{cite book |vauthors=Finkel R, Cubeddu LX, Clark MA |chapter=Chapter 41: Antiinfammatory Drugs |title=Pharmacology |date=2009 |publisher=Lippincott Williams & Wilkins |location=Philadelphia |isbn=978-0-7817-7155-9 |pages=499–518 (502) |edition=4th |chapter-url=https://books.google.com/books?id=Q4hG2gRhy7oC&pg=PA502 |access-date=14 April 2022 |archive-date=28 March 2023 |archive-url=https://web.archive.org/web/20230328005952/https://books.google.com/books?id=Q4hG2gRhy7oC&pg=PA502 |url-status=live }}</ref>

{{As of|2017|post=,}} an estimated 29% of approved drugs are enzyme inhibitors<ref name="Santos_2017" /> of which approximately one-fifth are [[kinase]] inhibitors.<ref name="Santos_2017">{{cite journal | vauthors = Santos R, Ursu O, Gaulton A, Bento AP, Donadi RS, Bologa CG, Karlsson A, Al-Lazikani B, Hersey A, Oprea TI, Overington JP | title = A comprehensive map of molecular drug targets | journal = Nature Reviews. Drug Discovery | volume = 16 | issue = 1 | pages = 19–34 (Figure 1C) | date = January 2017 | pmid = 27910877 | pmc = 6314433 | doi = 10.1038/nrd.2016.230 | quote = Figure 1C: Clinical success of privileged protein family classes (% approved drugs targeting each target class): Reductase 7.62, Kinase 5.94, Protease 3.35, Hydrolase 2.76, NPTase 2.09, Transferase 1.92, Lyase 1.59, Isomerase 1.51, Phosphodiesterase 1.50, Cytochrome p450 0.84, Epigenetic eraser 0.33, Total enzyme targets of approved drugs = 29.45% }}</ref> A notable class of kinase drug targets is the [[receptor tyrosine kinase]]s which are essential enzymes that regulate [[cell growth]]; their over-activation may result in cancer. Hence [[kinase inhibitor]]s such as [[imatinib]] are frequently used to treat malignancies.<ref name="Kannaiyan_2018">{{cite journal | vauthors = Kannaiyan R, Mahadevan D | title = A comprehensive review of protein kinase inhibitors for cancer therapy | journal = Expert Review of Anticancer Therapy | volume = 18 | issue = 12 | pages = 1249–1270 | date = December 2018 | pmid = 30259761 | pmc = 6322661 | doi = 10.1080/14737140.2018.1527688 }}</ref> [[Janus kinase]]s are another notable example of drug enzyme targets. [[Janus kinase inhibitor|Inhibitors of Janus kinases]] block the production of [[inflammatory cytokine]]s and hence these inhibitors are used to treat a variety of [[inflammatory disease]]s in including [[arthritis]], [[asthma]], and [[Crohn's disease]].<ref name="McLornan_2021">{{cite journal | vauthors = McLornan DP, Pope JE, Gotlib J, Harrison CN | title = Current and future status of JAK inhibitors | journal = Lancet | volume = 398 | issue = 10302 | pages = 803–816 | date = August 2021 | pmid = 34454676 | doi = 10.1016/S0140-6736(21)00438-4 | s2cid = 237311419 }}</ref>

An example of the structural similarity of some inhibitors to the substrates of the enzymes they target is seen in the figure comparing the drug [[methotrexate]] to [[folic acid]]. Folic acid is the oxidised form of the substrate of [[dihydrofolate reductase]], an enzyme that is potently inhibited by methotrexate. Methotrexate blocks the action of dihydrofolate reductase and thereby halts [[thymidine]] biosynthesis.<ref name = "Avendano_2015" /> This block of [[nucleotide]] biosynthesis is selectively toxic to rapidly growing cells, therefore methotrexate is often used in cancer chemotherapy.<ref>{{cite journal | vauthors = McGuire JJ | title = Anticancer antifolates: current status and future directions | journal = Current Pharmaceutical Design | volume = 9 | issue = 31 | pages = 2593–2613 | year = 2003 | pmid = 14529544 | doi = 10.2174/1381612033453712 }}</ref>

A common treatment for [[erectile dysfunction]] is [[sildenafil]] (Viagra).<ref name="common_2019">{{cite journal | vauthors = Goldstein I, Burnett AL, Rosen RC, Park PW, Stecher VJ | title = The Serendipitous Story of Sildenafil: An Unexpected Oral Therapy for Erectile Dysfunction | journal = Sexual Medicine Reviews | volume = 7 | issue = 1 | pages = 115–128 | date = January 2019 | pmid = 30301707 | doi = 10.1016/j.sxmr.2018.06.005 | s2cid = 52945888 }}</ref> This compound is a potent inhibitor of [[cGMP specific phosphodiesterase type 5]], the enzyme that degrades the signalling molecule [[cyclic guanosine monophosphate]].<ref>{{cite journal | vauthors = Maggi M, Filippi S, Ledda F, Magini A, Forti G | title = Erectile dysfunction: from biochemical pharmacology to advances in medical therapy | journal = European Journal of Endocrinology | volume = 143 | issue = 2 | pages = 143–154 | date = August 2000 | pmid = 10913932 | doi = 10.1530/eje.0.1430143 | doi-access = free }}</ref> This signalling molecule triggers smooth muscle relaxation and allows blood flow into the [[Corpus cavernosum penis|corpus cavernosum]], which causes an erection. Since the drug decreases the activity of the enzyme that halts the signal, it makes this signal last for a longer period of time.

====Antibiotics====
[[File:PDB 1PWC.png|thumb|alt=3D cartoon diagram of transpeptidase bound to penicillin G depicted as sticks|The structure of a complex between penicillin{{spaces}}G and the ''Streptomyces'' transpeptidase ({{Pdb|1PWC}})]]

Drugs are also used to inhibit enzymes needed for the survival of pathogens. For example, bacteria are surrounded by a thick [[Bacterial cell structure|cell wall]] made of a net-like polymer called [[peptidoglycan]]. Many [[antibiotic]]s such as [[penicillin]] and [[vancomycin]] inhibit the enzymes that produce and then cross-link the strands of this polymer together.<ref name="Cochrane_2020">{{cite journal | vauthors = Cochrane SA, Lohans CT | title = Breaking down the cell wall: Strategies for antibiotic discovery targeting bacterial transpeptidases | journal = European Journal of Medicinal Chemistry | volume = 194 | issue = | pages = 112262 | date = May 2020 | pmid = 32248005 | doi = 10.1016/j.ejmech.2020.112262 | s2cid = 214809706 | url = https://pure.qub.ac.uk/en/publications/breaking-down-the-cell-wall-strategies-for-antibiotic-discovery-targeting-bacterial-transpeptidases(e1bd1fbf-37a6-44ce-afdf-cc255bf217e6).html | access-date = 20 July 2022 | archive-date = 28 March 2023 | archive-url = https://web.archive.org/web/20230328005938/https://pure.qub.ac.uk/en/publications/breaking-down-the-cell-wall-strategies-for-antibiotic-discovery-t | url-status = live }}</ref><ref name="Buynak_2007">{{cite journal | vauthors = Buynak JD | title = Cutting and stitching: the cross-linking of peptidoglycan in the assembly of the bacterial cell wall | journal = ACS Chemical Biology | volume = 2 | issue = 9 | pages = 602–5 | date = September 2007 | pmid = 17894443 | doi = 10.1021/cb700182u }}</ref> This causes the cell wall to lose strength and the bacteria to burst. In the figure, a molecule of penicillin (shown in a ball-and-stick form) is shown bound to its target, the [[DD-Transpeptidase|transpeptidase]] from the bacteria ''Streptomyces'' R61 (the protein is shown as a [[ribbon diagram]]).

Antibiotic [[drug design]] is facilitated when an enzyme that is essential to the pathogen's survival is absent or very different in humans.<ref name="Dalhoff_2021">{{cite journal | vauthors = Dalhoff A | title = Selective toxicity of antibacterial agents-still a valid concept or do we miss chances and ignore risks? | journal = Infection | volume = 49 | issue = 1 | pages = 29–56 | date = February 2021 | pmid = 33367978 | pmc = 7851017 | doi = 10.1007/s15010-020-01536-y }}</ref> Humans do not make peptidoglycan, therefore antibiotics that inhibit this process are selectively toxic to bacteria.<ref>{{cite journal | vauthors = Mobley H | title = How do antibiotics kill bacterial cells but not human cells? | journal = Scientific American | date = 13 March 2006 | volume = 294 | issue = 6 | page = 98 | pmid = 16711368 | url = https://www.scientificamerican.com/article/how-do-antibiotics-kill-b/ | access-date = 9 April 2022 | archive-date = 9 April 2022 | archive-url = https://web.archive.org/web/20220409085735/https://www.scientificamerican.com/article/how-do-antibiotics-kill-b/ | url-status = live }}</ref> Selective toxicity is also produced in antibiotics by exploiting differences in the structure of the [[ribosome]]s in bacteria,<ref name="Zhang_2021">{{cite journal | vauthors = Zhang L, He J, Bai L, Ruan S, Yang T, Luo Y | title = Ribosome-targeting antibacterial agents: Advances, challenges, and opportunities | journal = Medicinal Research Reviews | volume = 41 | issue = 4 | pages = 1855–1889 | date = July 2021 | pmid = 33501747 | doi = 10.1002/med.21780 | s2cid = 231761270 }}</ref> or how they make [[fatty acid]]s.<ref name="Yao_2017">{{cite journal | vauthors = Yao J, Rock CO | title = Bacterial fatty acid metabolism in modern antibiotic discovery | journal = Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids | volume = 1862 | issue = 11 | pages = 1300–1309 | date = November 2017 | pmid = 27668701 | pmc = 5364071 | doi = 10.1016/j.bbalip.2016.09.014 }}</ref>

====Antivirals====
Drugs that inhibit enzymes needed for the [[Viral replication|replication of viruses]] are effective in treating viral infections.<ref>{{cite book | vauthors = Li G, Jing X, Pan, Zhang P, De Clercq E | chapter = Antiviral Classification | veditors = Bamford D, Zuckerman MA | title=Encyclopedia of Virology | edition = 4th | volume = 5 | publication-place=Amsterdam | publisher = Academic Press | date=2021 | isbn=978-0-12-814516-6 | oclc=1240584737 | pages= 129–130 | doi = 10.1016/B978-0-12-814515-9.00126-0 | doi-access = free }}</ref> [[Antiviral drug]]s include [[protease inhibitors]] used to treat [[HIV/AIDS]]<ref name="Voshavar_2019">{{cite journal | vauthors = Voshavar C | title = Protease Inhibitors for the Treatment of HIV/AIDS: Recent Advances and Future Challenges | journal = Current Topics in Medicinal Chemistry | volume = 19 | issue = 18 | pages = 1571–1598 | date = 2019 | pmid = 31237209 | doi = 10.2174/1568026619666190619115243 | s2cid = 195356119 }}</ref> and [[Hepatitis&nbsp;C]],<ref name="de_Leuw_2018">{{cite journal | vauthors = de Leuw P, Stephan C | title = Protease inhibitor therapy for hepatitis C virus-infection | journal = Expert Opinion on Pharmacotherapy | volume = 19 | issue = 6 | pages = 577–587 | date = April 2018 | pmid = 29595065 | doi = 10.1080/14656566.2018.1454428 | s2cid = 4489039 }}</ref> [[reverse-transcriptase inhibitor]]s targeting HIV/AIDS,<ref name="Li_2011">{{cite journal | vauthors = Li G, Wang Y, De Clercq E | title = Approved HIV reverse transcriptase inhibitors in the past decade | journal = Acta Pharmaceutica Sinica B | volume = 12 | issue = 4 | pages = 1567–1590 | date = April 2022 | pmid = 35847492 | pmc = 9279714 | doi = 10.1016/j.apsb.2021.11.009 }}</ref> [[neuraminidase inhibitor]]s targeting [[influenza]],<ref name="Gubareva_2022">{{cite journal | vauthors = Gubareva L, Mohan T | title = Antivirals Targeting the Neuraminidase | journal = Cold Spring Harbor Perspectives in Medicine | volume = 12 | issue = 1 | pages = a038455| date = January 2022 | pmid = 32152244 | doi = 10.1101/cshperspect.a038455 | pmc = 8725622 | s2cid = 212651676 }}</ref> and terminase inhibitors targeting [[human cytomegalovirus]].<ref name="Gentry_2019">{{cite journal | vauthors = Gentry BG, Bogner E, Drach JC | title = Targeting the terminase: An important step forward in the treatment and prophylaxis of human cytomegalovirus infections | journal = Antiviral Research | volume = 161 | issue = | pages = 116–124 | date = January 2019 | pmid = 30472161 | doi = 10.1016/j.antiviral.2018.11.005 | s2cid = 53763831 }}</ref>

===Pesticides===
Many [[pesticide]]s are enzyme inhibitors.<ref>{{cite book | vauthors = Kuhr RJ, Dorough HW |title=Carbamate Insecticides: Chemistry, Biochemistry, and Toxicology |date=1976 |publisher=CRC Press |location=Cleveland |isbn=978-0-87819-052-2}}</ref> [[Acetylcholinesterase]] (AChE) is an enzyme found in animals, from insects to humans. It is essential to nerve cell function through its mechanism of breaking down the neurotransmitter [[acetylcholine]] into its constituents, [[acetate]] and [[choline]].<ref>{{cite book | vauthors = Stone TW |title=CNS Neurotransmitters and Neuromodulators: Acetylcholine |date=October 2020 |publisher=CRC Press |location=Boca Raton |isbn=978-1-00-009898-3 |edition=1st}}</ref> This is somewhat unusual among neurotransmitters as most, including [[serotonin]], [[dopamine]], and [[norepinephrine]], are absorbed from the [[synaptic cleft]] rather than cleaved. A large number of AChE inhibitors are used in both medicine and agriculture.<ref name = "Gupta_2006">{{cite book |vauthors=Gupta RC |chapter=Classification and Uses of Organophosphates and Carbamates |veditors=Gupta RC |title=Toxicology of Organophosphate and Carbamate Compounds |date=2006 |publisher=Elsevier Academic Press |location=Amsterdam |isbn=978-0-08-054310-9 |pages=5–24 |chapter-url=https://books.google.com/books?id=mFFoWG-x4rAC&q=Classification+and+Uses+of+Organophosphates+and+Carbamates&pg=PA5 |access-date=21 July 2022 |archive-date=21 July 2022 |archive-url=https://web.archive.org/web/20220721073320/https://books.google.com/books?id=mFFoWG-x4rAC&q=Classification+and+Uses+of+Organophosphates+and+Carbamates&pg=PA5 |url-status=live }}</ref> Reversible competitive inhibitors, such as [[edrophonium]], [[physostigmine]], and [[neostigmine]], are used in the treatment of [[myasthenia gravis]]<ref name="Farmakidis_2018">{{cite journal | vauthors = Farmakidis C, Pasnoor M, Dimachkie MM, Barohn RJ | title = Treatment of Myasthenia Gravis | journal = Neurologic Clinics | volume = 36 | issue = 2 | pages = 311–337 | date = May 2018 | pmid = 29655452 | pmc = 6690491 | doi = 10.1016/j.ncl.2018.01.011 }}</ref> and in anaesthesia to reverse muscle blockade.<ref>{{cite book | chapter = Chapter 12. Cholinesterase Inhibitors & Other Pharmacologic Antagonists to Neuromuscular Blocking Agents. | veditors = Butterworth JF, IV, Mackey DC, Wasnick JD | title = Morgan & Mikhail's Clinical Anesthesiology | edition = 5th | publisher = McGraw Hill | date = 2013 | url = https://accessanesthesiology.mhmedical.com/content.aspx?bookid=564&sectionid=42800543 | access-date = 9 April 2022 | archive-date = 30 June 2022 | archive-url = https://web.archive.org/web/20220630044627/https://accessanesthesiology.mhmedical.com/content.aspx?bookid=564&sectionid=42800543 | url-status = live }}</ref> The [[carbamate]] pesticides are also examples of reversible AChE inhibitors. The [[organophosphate]] pesticides such as [[malathion]], [[parathion]], and [[chlorpyrifos]] irreversibly inhibit acetylcholinesterase.<ref name="Thapa_2017">{{cite journal | vauthors = Thapa S, Lv M, Xu H | title = Acetylcholinesterase: A Primary Target for Drugs and Insecticides | journal = Mini Reviews in Medicinal Chemistry | volume = 17 | issue = 17 | pages = 1665–1676 | date = 2017 | pmid = 28117022 | doi = 10.2174/1389557517666170120153930 }}</ref>

===Herbicides===
The herbicide [[glyphosate]] is an inhibitor of [[3-phosphoshikimate 1-carboxyvinyltransferase]],<ref name="pmid16547651">{{cite journal | vauthors = Tan S, Evans R, Singh B | title = Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops | journal = Amino Acids | volume = 30 | issue = 2 | pages = 195–204 | date = March 2006 | pmid = 16547651 | doi = 10.1007/s00726-005-0254-1 | s2cid = 2358278 }}</ref> other herbicides, such as the [[sulfonylurea]]s inhibit the enzyme [[acetolactate synthase]].<ref>{{cite book |vauthors=Hirai K, Uchida A, Ohno R |title=Herbicide Classes in Development |chapter=Major Synthetic Routes for Modern Herbicide Classes and Agrochemical Characteristics |quote=Chapter 10.2.1: Sulfonylurea acetolactate synthase inhibitors |veditors=Böger P, Wakabayashi K, Hirai K |date=2012 |publisher=Springer Science & Business Media |isbn=978-3-642-59416-8 |pages=179–195 |chapter-url=https://books.google.com/books?id=JSjzCAAAQBAJ&pg=PA180 |doi=10.1007/978-3-642-59416-8_10 |access-date=27 June 2022 |archive-date=27 June 2022 |archive-url=https://web.archive.org/web/20220627073108/https://books.google.com/books?id=JSjzCAAAQBAJ&pg=PA180 |url-status=live }}</ref> Both enzymes are needed for plants to make branched-chain [[amino acid]]s. Many other enzymes are inhibited by herbicides, including enzymes needed for the biosynthesis of [[lipid]]s and [[carotenoid]]s and the processes of [[photosynthesis]] and [[oxidative phosphorylation]].<ref>{{cite journal | vauthors = Duke SO | title = Overview of herbicide mechanisms of action | journal = Environmental Health Perspectives | volume = 87 | pages = 263–271 | date = July 1990 | pmid = 1980104 | pmc = 1567841 | doi = 10.2307/3431034 | jstor = 3431034 }}</ref>

== Discovery and design ==
[[File:Screening robotics for HTS-1-.jpg|thumb|alt=photo of robots at work|Robots are used for the high-throughput screening of chemical libraries to discover new enzyme inhibitors.]]

New drugs are the products of a long [[drug development]] process, the first step of which is often the discovery of a new enzyme inhibitor.<ref>{{cite book | vauthors = Ganguly AK, Alluri SS | chapter = Chapter 2: Enzyme Inhibitors |title=Medicinal Chemistry: A Look at How Drugs Are Discovered | date = 12 September 2021 |publisher=CRC Press |location=Boca Raton, Florida |isbn=978-1-00-043721-8 |pages=29–68}}</ref> There are two principle approaches of discovering these inhibitors.<ref name="Rufer_2021">{{cite journal | vauthors = Rufer AC | title = Drug discovery for enzymes | journal = Drug Discovery Today | volume = 26 | issue = 4 | pages = 875–886 | date = April 2021 | pmid = 33454380 | doi = 10.1016/j.drudis.2021.01.006 | s2cid = 231633284 | doi-access = free }}</ref>

The first general method is [[rational drug design]] based on mimicking the [[transition state]] of the chemical reaction catalysed by the enzyme.<ref>{{cite book | vauthors = Lindquist RN | chapter = The design of enzyme inhibitors: Transition state analogues. | veditors = Ariëns EJ | title = Drug Design: Medicinal Chemistry: A Series of Monographs | date = October 2013 | volume = 5 | pages = 24–80 | publisher = Elsevier }}</ref> The designed inhibitor often closely resembles the substrate, except that the portion of the substrate that undergoes chemical reaction is replaced by a chemically stable [[functional group]] that resembles the transition state. Since the enzyme has evolved to stabilise the transition state, [[transition state analogue]]s generally possess higher affinity for the enzyme compared to the substrate, and therefore are effective inhibitors.<ref name="Schramm_2018">{{cite journal | vauthors = Schramm VL | title = Enzymatic Transition States and Drug Design | journal = Chemical Reviews | volume = 118 | issue = 22 | pages = 11194–11258 | date = November 2018 | pmid = 30335982 | pmc = 6615489 | doi = 10.1021/acs.chemrev.8b00369 }}</ref>

The second way of discovering new enzyme inhibitors is [[high-throughput screening]] of large libraries of structurally diverse compounds to identify hit molecules that bind to the enzyme. This method has been extended to include [[virtual screening]] of databases of diverse molecules using computers,<ref>{{cite journal | vauthors = Scapin G | title = Structural biology and drug discovery | journal = Current Pharmaceutical Design | volume = 12 | issue = 17 | pages = 2087–2097 | year = 2006 | pmid = 16796557 | doi = 10.2174/138161206777585201 }}</ref><ref>{{cite journal | vauthors = Gohlke H, Klebe G | title = Approaches to the description and prediction of the binding affinity of small-molecule ligands to macromolecular receptors | journal = Angewandte Chemie | volume = 41 | issue = 15 | pages = 2644–2676 | date = August 2002 | pmid = 12203463 | doi = 10.1002/1521-3773(20020802)41:15<2644::AID-ANIE2644>3.0.CO;2-O }}</ref> which are then followed by experimental confirmation of binding of the virtual screening hits.<ref>{{cite journal | vauthors = Koppitz M, Eis K | title = Automated medicinal chemistry | journal = Drug Discovery Today | volume = 11 | issue = 11–12 | pages = 561–568 | date = June 2006 | pmid = 16713909 | doi = 10.1016/j.drudis.2006.04.005 }}</ref> Complementary approaches that can provide new starting points for inhibitors include [[fragment-based lead discovery]]<ref name="Ciulli_2007">{{cite journal | vauthors = Ciulli A, Abell C | title = Fragment-based approaches to enzyme inhibition | journal = Current Opinion in Biotechnology | volume = 18 | issue = 6 | pages = 489–96 | date = December 2007 | pmid = 17959370 | pmc = 4441723 | doi = 10.1016/j.copbio.2007.09.003 }}</ref> and [[DNA Encoded Chemical Libraries]] (DEL).<ref name="Satz_2021">{{cite journal | vauthors = Satz AL, Kuai L, Peng X | title = Selections and screenings of DNA-encoded chemical libraries against enzyme and cellular targets | journal = Bioorganic & Medicinal Chemistry Letters | volume = 39 | issue = | pages = 127851 | date = May 2021 | pmid = 33631371 | doi = 10.1016/j.bmcl.2021.127851 | s2cid = 232059044 | doi-access = free }}</ref>

Hits from any of the above approaches can be [[Hit to lead|optimised]] to high affinity binders that efficiently inhibit the enzyme.<ref>{{cite book | vauthors = Perez O, Pena J, Fernandez-Vega V, Scampavia L, Spicer T | chapter = Chapter 4: High Throughput Screening | pages = 47–69 |title=Drug Discovery and Development |date=2019 |publisher=CRC Press |location=Boca Raton, Florida |isbn=978-1-315-11347-0 }}</ref> [[Drug design|Computer-based methods]] for predicting the binding orientation and affinity of an inhibitor for an enzyme such as [[molecular docking]]<ref name="Scarpino_2020">{{cite journal | vauthors = Scarpino A, Ferenczy GG, Keserű GM | title = Covalent Docking in Drug Discovery: Scope and Limitations | journal = Current Pharmaceutical Design | volume = 26 | issue = 44 | pages = 5684–5699 | date = 2020 | pmid = 33155894 | doi = 10.2174/1381612824999201105164942 | s2cid = 226271153 }}</ref> and [[molecular mechanics]] can be used to assist in the optimisation process.<ref>{{cite journal | vauthors = Elsässer B, Goettig P | title = Mechanisms of Proteolytic Enzymes and Their Inhibition in QM/MM Studies | journal = International Journal of Molecular Sciences | volume = 22 | issue = 6 | pages = 3232 | date = March 2021 | pmid = 33810118 | pmc = 8004986 | doi = 10.3390/ijms22063232 | doi-access = free }}</ref> New inhibitors are used to obtain [[X-ray crystallography#Biological macromolecular crystallography|crystallographic structures]] of the enzyme in an inhibitor/enzyme complex to show how the molecule is binding to the active site, allowing changes to be made to the inhibitor to optimise binding in a process known as [[Drug design#Structure-based|structure-based drug design]].<ref name = "Copeland_2013" />{{rp|66}} This test and improve cycle is repeated until a sufficiently potent inhibitor is produced.

==See also==
{{Portal|Biology}}
*[[Activity-based proteomics]] – a branch of [[proteomics]] that uses covalent enzyme inhibitors as reporters to monitor enzyme activity.
*[[Antimetabolite]] – an enzyme inhibitor that is used to interfere with cell growth and division
*[[Transition state analogue]] – a type of enzyme inhibitor that mimics the transition state of the chemical reaction catalysed by the enzyme

==References==
{{Reflist}}

==External links==
*{{cite web | title = BRENDA | url = https://www.brenda-enzymes.org | archive-url = https://web.archive.org/web/20220401024216/https://www.brenda-enzymes.org/ | archive-date= 1 April 2022| url-status = live }}, Database of enzymes giving lists of known inhibitors for each entry
*{{cite web | title = PubChem | url = https://pubchem.ncbi.nlm.nih.gov/ | work = National Center for Biotechnology Information | publisher = National Library of Medicine }} Database of drugs and enzyme inhibitors
*{{cite web | title = Symbolism and Terminology in Enzyme Kinetics | url = https://iubmb.qmul.ac.uk/kinetics/ek4t6.html#p6 | archive-url = https://web.archive.org/web/20060620032006/http://www.chem.qmul.ac.uk/iubmb/kinetics/ek4t6.html#p6 | archive-date = 20 June 2006 }} Recommendations of the Nomenclature Committee of the International Union of Biochemistry (NC-IUB) on enzyme inhibition terminology

{{Enzyme inhibition}}
{{Medicinal chemistry}}
{{Enzymes}}
{{Authority control}}

[[Category:Biochemical reactions]]
[[Category:Medicinal chemistry]]
[[Category:Metabolism]]
[[Category:Enzyme inhibitors| ]]