A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors (Short Communication)

Cornish‐Bowden, Athel

doi:10.1042/bj1370143

Cited by 900 publications

(492 citation statements)

References 3 publications

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“…3) revealed the non-competitive inhibition [21] of the levansucrase-catalysed reaction when using melibiose with an inhibitory constant of K i = 54 mM. In addition, differences between the enzymes from various sources were observed, since melibiose did not affect the activity of Z. mobilis levansucrase from a recombinant E. coli [7].…”

Section: Resultsmentioning

confidence: 99%

Formation of Levan from Raffinose by Levansucrase of Zymomonas mobilis

Andersone

Auzina

Vigants

et al. 2004

Engineering in Life Sciences

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Levansucrase (EC 2.4.1.10.) of Zymomonas mobilis 113S can perform the polymerisation of fructose moiety from raffinose to levan concomitantly with a release of non-catabolised melibiose into the medium. The kinetic parameters of the levansucrase-catalysed reaction provide even higher reaction velocities on raffinose as compared to sucrose, particularly at low substrate concentrations. A decreased value in the number of the average molecular mass (M n = 1693 kDa), an increased intrinsic viscosity (g = 49.47 cm 3 /g), and a diminished Huggin's constant (K ' = 0.67) are intrinsic to the levan synthesis from raffinose, indicating certain structural peculiarities compared to a polysaccharide obtained from sucrose (M n = 1851 kDa, [g] = 42.47 cm 3 /g, K' = 1.21).

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Section: Resultsmentioning

confidence: 99%

Formation of Levan from Raffinose by Levansucrase of Zymomonas mobilis

Andersone

Auzina

Vigants

et al. 2004

Engineering in Life Sciences

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“…The plots were assessed by a weighted least square analysis that assumed the variance of ν to be a constant percentage of ν for the entire data set. To confirm the mode of inhibition, CornishBowden plots were obtained by plotting S/v (substrate/velocity ratio) versus inhibitor concentration [21]. Data analysis was performed with GraphPad Prism 4.03 software (GraphPad Software Inc.).…”

Section: Kinetic Inhibition Studiesmentioning

confidence: 99%

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: Synthesis, pharmacological evaluation and mechanistic studies

Pietro

Viayna

Vicente‐García

et al. 2014

European Journal of Medicinal Chemistry

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[1,6]naphthyridines differently substituted at positions 1, 5, and 9 have been designed from the pyrano[3,2-c]quinoline derivative 1, a weak inhibitor of acetylcholinesterase (AChE) with predicted ability to bind to the AChE peripheral anionic site (PAS), at the entrance of the catalytic gorge. Fourteen novel benzonaphthyridines have been synthesized through synthetic sequences involving as the key step a multicomponent Povarov reaction between an aldehyde, an aniline and an enamine or an enamide as the activated alkene. The novel compounds have been tested against Electrophorus electricus AChE (EeAChE), human recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling. We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217-and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities. recombinant AChE (hAChE), and human serum butyrylcholinesterase (hBChE), and their brain penetration has been assessed using the PAMPA-BBB assay. Also, the mechanism of AChE inhibition of the most potent compounds has been thoroughly studied by kinetic studies, a propidium displacement assay, and molecular modelling.We have found that a seemingly small structural change such as a double O → NH bioisosteric replacement from the hit 1 to 16a results in a dramatic increase of EeAChE and hAChE inhibitory activities (>217-and >154-fold, respectively), and in a notable increase in hBChE inhibitory activity (> 11-fold), as well. An optimized binding at the PAS besides additional interactions with AChE midgorge residues seem to account for the high hAChE inhibitory potency of 16a (IC 50 = 65 nM), which emerges as an interesting anti-Alzheimer lead compound with potent dual AChE and BChE inhibitory activities.

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“…Quantification of substrate reaction product (phosphate) was performed colorimetrically by the ammonium molybdate method. Inhibitor constants were obtained from averages of Dixon and s/v plots (29,30).…”

Section: Methodsmentioning

confidence: 99%

Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity

Addison

Azari

Sørensen

et al. 2007

Journal of Biological Chemistry

334

267

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Inorganic pyrophosphate (PP i ) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P i in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP i are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP i on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP i . This inhibition could be reversed by Tnap, but not if PP i was bound to mineral. PP i also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP i was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP i , hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP i inhibits Tnap-mediated P i release from ␤-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP i prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

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A simple graphical method for determining the inhibition constants of mixed, uncompetitive and non-competitive inhibitors (Short Communication)

Abstract: A new method is described for plotting kinetic results for inhibited enzyme-catalysed reactions. It provides a simple way of determining the inhibition constant, K'(i), of an uncompetitive, mixed or non-competitive inhibitor.

Cited by 900 publications

References 3 publications

Formation of Levan from Raffinose by Levansucrase of Zymomonas mobilis

Formation of Levan from Raffinose by Levansucrase of Zymomonas mobilis

1,2,3,4-Tetrahydrobenzo[h][1,6]naphthyridines as a new family of potent peripheral-to-midgorge-site inhibitors of acetylcholinesterase: Synthesis, pharmacological evaluation and mechanistic studies

Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity

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