Mixed inhibition

Last updated September 11, 2021

Mixed inhibition is a type of enzyme inhibition in which the inhibitor may bind to the enzyme whether or not the enzyme has already bound the substrate but has a greater affinity for one state or the other.^[1] It is called "mixed" because it can be seen as a conceptual "mixture" of competitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has not already bound, and uncompetitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has already bound. If the ability of the inhibitor to bind the enzyme is exactly the same whether or not the enzyme has already bound the substrate, it is known as a non-competitive inhibitor.^[1]^[2] Non-competitive inhibition is sometimes thought of as a special case of mixed inhibition.

In mixed inhibition, the inhibitor binds to an allosteric site, i.e. a site different from the active site where the substrate binds. However, not all inhibitors that bind at allosteric sites are mixed inhibitors. ^[1]

Mixed inhibition may result in either:

A decrease in the apparent affinity of the enzyme for the substrate (Km value appears to increase; $K_{m}^{\text{app}}>K_{m}$ ) -- seen in cases where the inhibitor favours binding to the free enzyme. More closely mimics competitive binding.
An increase in the apparent affinity of the enzyme for the substrate (Km value appears to decrease; $K_{m}^{\text{app}}<K_{m}$ ) -- seen in cases where the inhibitor favours binding to the enzyme-substrate complex. More closely mimics uncompetitive binding.

In either case the inhibition decreases the apparent maximum enzyme reaction rate ( $V_{max}^{\text{app}}<V_{max}$ ).^[3]

Mathematically, mixed inhibition occurs when the factors α and α’ (introduced into the Michaelis-Menten equation to account for competitive and uncompetitive inhibition, respectively) are both greater than 1.

In the special case where α = α’, noncompetitive inhibition occurs, in which case $V_{max}^{app}$ is reduced but $K_{m}$ is unaffected. This is very unusual in practice.^[3]

Biological examples

In gluconeogenesis, the enzyme cPEPCK (cystolic phosphoenolpyruvate carboxykinase) is responsible for converting oxaloacetate into phosphoenolpyruvic acid, or PEP, when guanosine triphosphate, GTP, is present. This step is exclusive for gluconeogenesis, which occurs under fasting condition's due to the body's depletion of glucose. cPEPCK is known to be regulated by Genistein, an isoflavone that is naturally found in a number of plants. ^[4] It was first proven that genistein inhibits the activity of cPEPCK. In a study, the presence of this isoflavone resulted in a decrease in the level of blood sugar. A lowered blood sugar level means less glucose is in the blood. If this occurs in a subject that is fasting, this is because the gluconeogenesis was inhibited, preventing increased production of glucose. The ability of genistein to lower a person's blood sugar level allows it to be referred to as an anti-diabetic property. ^[4] The mechanism in which genistein inhibited the enzyme cPEPCK was further evaluated. First, cPEPCK was placed in the presence of 3-Mercaptopropionic acid, or 3-MPA, a known inhibitor of the enzyme. It was compared to the results of placing cPEPCK in the presence of genistein, which revealed that the mechanism of mixed inhibition was used to decrease cPEPCK's activity. ^[4] cPEPCK undergoes multiple configurations when catalyzing the formation of PEP. It can be either unbound, bound to GDP or bound to GTP. An experiment that studied the affinity for genistein in these different configurations was conducted. It revealed that geinstein favors binding to the cPEPCK with a bound GTP than then the enzyme with a bound GDP, which was found to be less stable.^[4] This was because the GTP-bound cPEPCK revealed an extended binding site for genistein.^[4] This is the same binding site as the enzyme's intended substrate, oxaloacetate while the other configurations did not do so in the presence of genistein. ^[4] This provided evidence that the mechanism of inhibition of cPEPCK by genistein was a mixture of competitive and non-competitive inhibition.

A kallikrein is a type of serine protease, which cleaves peptide bonds after certain amino acids in a protein. These 15 kallikreins, KLK1 to KLK15, are found in human tissues. The ability for this molecule to cleave proteins results in the effective activation of cell surface receptors, making them crucial elements of many biological signal transduction pathways, and its amplification through cascades. This family of serine proteases is often a biomarker to diseases, and therefore, have become a target for inhibition. ^[5] Inhibition of these kallikreins results in possible therapy for diseases such as metastatic cancer or Alzheimer's disease. ^[5] Fukugetin, or (+)-morelloflavone, is a type of plant biflavonoid isolated from Garcinia brasiliensis. ^[5] After isolating fukugetin, it was placed with KLK1, KLK2, KLK3, KLK4, KLK5, KLK6, and KLK7 in varying concentrations.^[5] This allowed for the analysis of enzyme kinetics through derivation of parameters Km and Vmax. Through the model of Michaelis-Menten kinetics, the Eadie-Hofstee diagram was plotted.^[5] It confirmed that fukugetin acts as a mixed inhibitor by exhibiting varying but present affinities for the enzyme alone and the enzyme-substrate complex. Analyzing through kinetics, fukugetin decreased the Vmax while it increased the Km for these KLKs.^[5] Typically, in competitive inhibition, Vmax remains the same while Km increases, and in non-competitive inhibition, Vmax decreases while Km remains the same. The change in both of these variables is another finding consistent with the effects of a mixed inhibitor.

Related Research Articles

Enzymes are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. Almost all metabolic processes in the cell need enzyme catalysis in order to occur at rates fast enough to sustain life. Metabolic pathways depend upon enzymes to catalyze individual steps. The study of enzymes is called enzymology and a new field of pseudoenzyme analysis has recently grown up, recognising that during evolution, some enzymes have lost the ability to carry out biological catalysis, which is often reflected in their amino acid sequences and unusual 'pseudocatalytic' properties.

In biochemistry, allosteric regulation is the regulation of an enzyme by binding an effector molecule at a site other than the enzyme's active site.

Pyruvate kinase is the enzyme involved in the last step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP. Pyruvate kinase was inappropriately named before it was recognized that it did not directly catalyze phosphorylation of pyruvate, which does not occur under physiological conditions. Pyruvate kinase is present in four distinct, tissue-specific isozymes in animals, each consisting of particular kinetic properties necessary to accommodate the variations in metabolic requirements of diverse tissues.

In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may include other proteins, enzyme substrates, second messengers, hormones, or allosteric modulators. The binding event is often, but not always, accompanied by a conformational change that alters the protein's function. Binding to protein binding sites is most often reversible, but can also be covalent reversible or irreversible.

In biochemistry, the Lineweaver–Burk plot is a graphical representation of the Lineweaver–Burk equation of enzyme kinetics, described by Hans Lineweaver and Dean Burk in 1934. The Lineweaver–Burk plot for inhibited enzymes can be compared to no inhibitor to determine how the inhibitor is competing with the enzyme.

In biochemistry and pharmacology, receptors are chemical structures, composed of protein, that receive and transduce signals that may be integrated into biological systems. These signals are typically chemical messengers which bind to a receptor and cause some form of cellular/tissue response, e.g. a change in the electrical activity of a cell. There are three main ways the action of the receptor can be classified: relay of signal, amplification, or integration. Relaying sends the signal onward, amplification increases the effect of a single ligand, and integration allows the signal to be incorporated into another biochemical pathway.

A receptor antagonist is a type of receptor ligand or drug that blocks or dampens a biological response by binding to and blocking a receptor rather than activating it like an agonist. Antagonist drugs interfere in the natural operation of receptor proteins. They are sometimes called blockers; examples include alpha blockers, beta blockers, and calcium channel blockers. In pharmacology, antagonists have affinity but no efficacy for their cognate receptors, and binding will disrupt the interaction and inhibit the function of an agonist or inverse agonist at receptors. Antagonists mediate their effects by binding to the active site or to the allosteric site on a receptor, or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist–receptor complex, which, in turn, depends on the nature of antagonist–receptor binding. The majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally defined binding sites on receptors.

Glutamate dehydrogenase is an enzyme observed in both prokaryotes and eukaryotic mitochondria. The aforementioned reaction also yields ammonia, which in eukaryotes is canonically processed as a substrate in the urea cycle. Typically, the α-ketoglutarate to glutamate reaction does not occur in mammals, as glutamate dehydrogenase equilibrium favours the production of ammonia and α-ketoglutarate. Glutamate dehydrogenase also has a very low affinity for ammonia, and therefore toxic levels of ammonia would have to be present in the body for the reverse reaction to proceed. However, in brain, the NAD+/NADH ratio in brain mitochondria encourages oxidative deamination. In bacteria, the ammonia is assimilated to amino acids via glutamate and aminotransferases. In plants, the enzyme can work in either direction depending on environment and stress. Transgenic plants expressing microbial GLDHs are improved in tolerance to herbicide, water deficit, and pathogen infections. They are more nutritionally valuable.

The half maximal inhibitory concentration (IC₅₀) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC₅₀ is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%. The biological component could be an enzyme, cell, cell receptor or microorganism. IC₅₀ values are typically expressed as molar concentration.

Non-competitive inhibition is a type of enzyme inhibition where the inhibitor reduces the activity of the enzyme and binds equally well to the enzyme whether or not it has already bound the substrate.

Enzyme kinetics is the study of the rates of enzyme-catalysed chemical reactions. In enzyme kinetics, the reaction rate is measured and the effects of varying the conditions of the reaction are investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism, how its activity is controlled, and how a drug or a modifier might affect the rate.

Uncompetitive inhibition, also known as anti-competitive inhibition, takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate. Uncompetitive inhibition typically occurs in reactions with two or more substrates or products.

An enzyme inhibitor is a molecule that binds to an enzyme and decreases its activity. By binding to enzymes' active sites, inhibitors reduce the compatibility of substrate and enzyme and this leads to the inhibition of Enzyme-Substrate complexes' formation, preventing the catalysis of reactions and decreasing the amount of product produced by a reaction. It can be said that as the concentration of enzyme inhibitors increases, the rate of enzyme activity decreases, and thus, the amount of product produced is inversely proportional to the concentration of inhibitor molecules. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used in pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity, while enzyme substrates bind and are converted to products in the normal catalytic cycle of the enzyme.

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Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme in the lyase family used in the metabolic pathway of gluconeogenesis. It converts oxaloacetate into phosphoenolpyruvate and carbon dioxide.

Bithionol is an antibacterial, anthelmintic, and algaecide. It is used to treat Anoplocephala perfoliata (tapeworms) in horses and Fasciola hepatica.

Substrate analogs, are chemical compounds with a chemical structure that resemble the substrate molecule in an enzyme-catalyzed chemical reaction. Substrate analogs can act as competitive inhibitors of an enzymatic reaction. An example is phosphoramidate to the Tetrahymena group I ribozyme

In molecular biology, cyanase is an enzyme which catalyses the bicarbonate dependent metabolism of cyanate to produce ammonia and carbon dioxide. The systematic name of this enzyme is carbamate hydrolyase. In E. coli, cyanase is an inducible enzyme and is encoded for by the cynS gene. Cyanate is a toxic anion, and cyanase catalyzes the metabolism into the benign products of carbon dioxide and ammonia.

Competitive inhibition Interruption of a chemical pathway

Competitive inhibition is interruption of a chemical pathway owing to one chemical substance inhibiting the effect of another by competing with it for binding or bonding. Any metabolic or chemical messenger system can potentially be affected by this principle, but several classes of competitive inhibition are especially important in biochemistry and medicine, including the competitive form of enzyme inhibition, the competitive form of receptor antagonism, the competitive form of antimetabolite activity, and the competitive form of poisoning.

Substrate inhibition in bioreactors occurs when the concentration of substrate exceeds the optimal parameters and reduces the growth rate of the cells within the bioreactor. This is often confused with substrate limitation, which describes environments in which cell growth is limited due to of low substrate. Limited conditions can be modeled with the Monod equation; however, the Monod equation is no longer suitable in substrate inhibiting conditions. A Monod deviation, such as the Haldane (Andrew) equation, is more suitable for substrate inhibiting conditions. These cell growth models are analogous to equations that describe enzyme kinetics, although, unlike enzyme kinetics parameters, cell growth parameters are generally empirically estimated.

References

1 2 3 "Types of Inhibition". Archived from the original on 8 September 2011. Retrieved 2 April 2012.
↑ "Enzyme inhibition". London South Bank University. Archived from the original on 19 March 2012. Retrieved 2 April 2012.
1 2 Storey, Kenneth B. (2004). Functional Metabolism: Regulation and Adaptation. Wiley-IEEE. p. 12. ISBN 978-0-471-41090-4.
1 2 3 4 5 6 Katiyar, Shashank Prakash (2015). "Mixed Inhibition cPEPCK by Geinstein, Using an Extended Binding Site Located Adjacent to Its Catalytic Cleft". PLOS ONE. 10 (11): e0141987. doi: 10.1371/journal.pone.0141987 . PMC 4631375 . PMID 26528723 – via NCBI.
1 2 3 4 5 6 Santos, Jorge A. N. (2016). "The Natural Flavone Fukugetin as a Mixed-Type Inhibitor for Human Tissue Kallikreins". Bioorganic & Medicinal Chemistry Letters. 26 (5): 1485–9. doi:10.1016/j.bmcl.2016.01.039. PMID 26848109.

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[NIH_Inhibition-1] 1 2 3 "Types of Inhibition". Archived from the original on 8 September 2011. Retrieved 2 April 2012.

[2] "Enzyme inhibition". London South Bank University. Archived from the original on 19 March 2012. Retrieved 2 April 2012.

[funmet-3] 1 2 Storey, Kenneth B. (2004). Functional Metabolism: Regulation and Adaptation. Wiley-IEEE. p. 12. ISBN 978-0-471-41090-4.

[:0-4] 1 2 3 4 5 6 Katiyar, Shashank Prakash (2015). "Mixed Inhibition cPEPCK by Geinstein, Using an Extended Binding Site Located Adjacent to Its Catalytic Cleft". PLOS ONE. 10 (11): e0141987. doi: 10.1371/journal.pone.0141987 . PMC 4631375 . PMID 26528723 – via NCBI.

[:1-5] 1 2 3 4 5 6 Santos, Jorge A. N. (2016). "The Natural Flavone Fukugetin as a Mixed-Type Inhibitor for Human Tissue Kallikreins". Bioorganic & Medicinal Chemistry Letters. 26 (5): 1485–9. doi:10.1016/j.bmcl.2016.01.039. PMID 26848109.

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