the enzyme. instead, kobs/ values are used, where kobs is the observed pseudo-first order rate of inactivation (obtained by plotting the log of % activity versus time) and is the concentration of inhibitor. the kobs/ parameter is valid as long as the inhibitor does not saturate binding with the enzyme (in which case kobs = kinact) where kinact is the rate of inactivation. measuring irreversible inhibition mechanismkinetic 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 kinact. 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 kinact and ki. 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. this is usually done using a maldi-tof mass spectrometer. 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 peptides 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. slow binding 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. 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. 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, allopurinol, and the activated form of acyclovir. some examples trypanothione reductase with the lower molecule of an inhibitor bound irreversibly and the upper one reversibly. created from bond et al, 2004. (pdb: 1gxf​) diisopropylfluorophosphate (dfp) is an example of an irreversible 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 active site, deactivating it. 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 mg. suicide inhibition is an unusual type of irreversible inhibition where the enzyme converts the inhibitor into a reactive form in its active site. an example is the inhibitor of polyamine biosynthesis, 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. since irreversible inhibition often involves the initial formation of a non-covalent enzyme inhibitor (ei) complex, it is sometimes possible for an inhibitor to bind to an enzyme in more than one way. for example, in the figure showing 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. applications enzyme inhibitors are found in nature and also produced artificially in the laboratory. naturally occurring enzyme inhibitors regulate many metabolic processes and are essential for life. in addition, naturally produced poisons are often enzyme inhibitors that have evolved for use as toxic agents against predators, prey, and competing organisms. these natural toxins include some of the most poisonous substances known. artificial inhibitors are often used as drugs, but can also be insecticides such as malathion, herbicides such as glyphosate, or disinfectants such as triclosan. other artificial enzyme inhibitors block acetylcholinesterase, an enzyme which breaks down acetylcholine, and are used as nerve agents in chemical warfare. metabolic regulation enzyme inhibition is a common feature of metabolic pathway control in cells. 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 glycolytic pathway is a classic example. this catabolic pathway consumes glucose and produces atp, nadh and pyruvate. a key step for the regulation of glycolysis is an early reaction in the pathway catalysed by phosphofructokinase‑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 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 adp are examples of metabolites that are allosteric activators. 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 zymogens. 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. 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. other examples of physiological enzyme inhibitor proteins include the barstar inhibitor of the bacterial ribonuclease barnase. natural poisons to discourage seed predation, legumes contain trypsin inhibitors that interfere with digestion. animals and plants have evolved to synthesise a vast array of poisonous products including secondary metabolites, peptides and proteins that can act as inhibitors. natural toxins are usually small organic molecules and are so diverse that there are probably natural inhibitors for most metabolic processes. 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 pacific yew tree, binds tightly to tubulin dimers and inhibits their assembly into microtubules in the cytoskeleton. many natural poisons act as neurotoxins that can cause paralysis leading to death and function for defence against predators or in hunting and capturing prey. some of these natural inhibitors, despite their toxic attributes, are valuable for therapeutic uses at lower doses. an example of a neurotoxin are the glycoalkaloids, from the plant species in the family solanaceae (includes potato, tomato and eggplant), that are acetylcholinesterase inhibitors. 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 muscarinic acetylcholine receptors. 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 ii enzyme from transcribing dna. the algal toxin microcystin is also a peptide and is an inhibitor of protein phosphatases. this toxin can contaminate water supplies after algal blooms and is a known carcinogen that can also cause acute liver haemorrhage and death at higher doses. proteins can also be natural poisons or antinutrients, such as the trypsin inhibitors (discussed in the "metabolic regulation" section above) that are found in some legumes. 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 beans. this enzyme is a glycosidase that inactivates ribosomes. since ricin is a catalytic irreversible inhibitor, this allows just a single molecule of ricin to kill a cell. drugs the coenzyme folic acid (top) compared to the anti-cancer drug methotrexate (bottom) 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. this inhibition in turn suppresses the production of proinflammatory prostaglandins and thus aspirin may be used to reduce pain, fever, and inflammation. as of 2017, an estimated 29% of approved drugs are enzyme inhibitors of which approximately one-fifth are kinase inhibitors. a notable class of kinase drug targets is the receptor tyrosine kinases which are essential enzymes that regulate cell growth; their over-activation may result in cancer. hence kinase inhibitors such as imatinib are frequently used to treat malignancies. janus kinases are another notable example of drug enzyme targets. inhibitors of janus kinases block the production of inflammatory cytokines and hence these inhibitors are used to treat a variety of inflammatory diseases in including arthritis, asthma, and crohn's disease. 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. this block of nucleotide biosynthesis is selectively toxic to rapidly growing cells, therefore methotrexate is often used in cancer chemotherapy. a common treatment for erectile dysfunction is sildenafil (viagra). this compound is a potent inhibitor of cgmp specific phosphodiesterase type 5, the enzyme that degrades the signalling molecule cyclic guanosine monophosphate. this signalling molecule triggers smooth muscle relaxation and allows blood flow into the 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 the structure of a complex between penicillin 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 cell wall made of a net-like polymer called peptidoglycan. many antibiotics such as penicillin and vancomycin inhibit the enzymes that produce and then cross-link the strands of this polymer together. this causes the cell wall to lose strength and the bacteria to burst. in the figure, a molecule of