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Enzyme Kinetics and Drugs as Enzyme Inhibitors
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
The characteristic Lineweaver–Burk plot is composed of parallel lines with a slope value given by KM/vmax being constant for all concentrations of an uncompetitive inhibitor.
Identification Of Receptors In Vitro
Published in William C. Eckelman, Lelio G. Colombetti, Receptor-Binding Radiotracers, 2019
The difference between this expression and Equation 2 (the same situation in the absence of inhibitor) is that the Kd is multiplied by the factor (1 + [I]/Ki)· Since that factor is always greater than or equal to 1, the effect is to raise the apparent Kd. In an experiment where [I] is held constant, and [L] is varied, a straight line is therefore observed on Lineweaver-Burk or Scatchard plots, with the apparent Kd equal to Kd (1 + [I]/Ki). This allows the calculation of Ki·, if Kd is known. Figure 5 shows the expected result on Scatchard and Lineweaver-Burk plots. Since the Bmax is not affected, the y-intercept of the Lineweaver-Burk plot, and the x-intercept of the Scatchard plot, are unchanged. When that is true, it is a good indication that inhibition is competitive, and that Equation 13 is followed.
The Study of Drug Metabolism Using Radiotracers
Published in Graham Lappin, Simon Temple, Radiotracers in Drug Development, 2006
At its simplest level, the radiochromatographic profiles are directly compared between species to determine whether any species-specific metabolites are formed, particularly in humans. In addition to a simple profile, the enzymology and kinetics can be studied. In particular, it is important to understand which CYP P450 enzymes are involved in the metabolism of the drug, as this provides a better understanding of potential drug-drug interactions (Section 3.4.13) and polymorphisms (Section 3.4.15). In these experiments the turnover of the test drug in vitro should be limited to <20%, which is achieved by varying the incubation time and protein concentration. Once the major metabolites are established, incubation conditions are optimized to ensure linear kinetics, in terms of incubation time and enzyme concentration. The kinetic parameters (km and Vmax) governing the formation of each metabolite of interest may then be determined. Incubations are typically performed at eight or more drug concentrations, over a logarithmic scale. The data are plotted in a linear transform to determine Kmand Vmax, such as 1/rate against 1/substrate concentration (Lineweaver-Burk plot) or rate against rate/substrate concentration (Eadie-Hofstee plot).
Curcumin analogues exert potent inhibition on human and rat gonadal 3β-hydroxysteroid dehydrogenases as potential therapeutic agents: structure-activity relationship and in silico docking
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Xinyi Qiao, Lei Ye, Jialin Lu, Chengshuang Pan, Qianjin Fei, Yang Zhu, Huitao Li, Han Lin, Ren-shan Ge, Yiyan Wang
Enzyme parameter analysis was done by GraphPad (version 8.0, GraphPad Software, San Diego, CA, USA) as previously described18. ① Time curve of reaction: linear regression; ② Michaelis-Menten constant (Km) and Vmax was calculated by a nonlinear regression using equation: V = Vmax*[P5]/(Km + [P5]), where V is velocity (pmol/mg/min) and [P5] is substrate concentration (µM); ③ Effect of 100 µM curcumin analogues on 3β-HSD: Residual activity related to DMSO control (100%) was calculated; ④ IC50 value was calculated by a nonlinear regression of [inhibitor] vs. response (three parameters); ⑤ mode of action vs. P5 was determined by a nonlinear regression in enzyme kinetics – inhibition (mixed model) for parameters of “α” and Ki by following equations: V = VmaxApp*[P5]/(KmApp + [P5]), where [P5] was substrate concentration, V was the enzyme activity; and VmaxApp and KmApp were derived from VmaxApp = Vmax/(1 + I/(α*Ki)) and KmApp = Km*(1 + I/Ki)/(1 + I/(α*Ki)), where I was inhibitor’s concentration; “α” was determination factor for mode of action. When “α = 1”, it was a non-competitive inhibitor; when “α > 1 or <1", it was a mixed inhibitor; when “α was very small but larger than zero", it was an uncompetitive inhibitor; when “α was very large", it was a competitive inhibitor. ⑥ Lineweaver-Burk plot was drawn using linear regression.
Bromhexine and its inhibitory effect on lipase – kinetics and structural study
Published in Archives of Physiology and Biochemistry, 2022
Asma Gholami, Dariush Minai-Tehrani, Fereshteh Eftekhar
The buffer for enzyme assay was prepared by adding different concentrations (0.06–0.5 mM) of pNPP (soluble in isopropanol) as the substrate to 0.1 M Tris buffer pH 8 containing 0.1% (vol/vol) Tween 80. The reaction was started by adding 20 µL of the supernatant to the working buffer in the test tube. Final volume in the test tube was always 2 mL. The lipase catalytic activity was continuously checked by following the increased yellow color of the product (p-nitrophenol) with absorption at 410 nm (Talebi et al. 2018). The enzyme assay was detected in the absence and presence of bromhexine with concentrations 0.048–0.32 mM. The assay was monitored for 10 min; this time was enough for the enzyme to reach the plateau in the progress curve (substrate depletion). The extinction coefficient of the product, p-nitrophenol (18.8 × 103 mol–1 cm–1), was used to determine its concentration and enzyme activity. Lineweaver–Burk plot was applied to obtain the kinetic parameters of the enzyme and type of inhibition. All the assays were carried out at room temperature (25–28 °C) using a Perkin-Elmer visible spectrophotometer. Lowry method was used for protein determination. All the assays were repeated as three separated experiments.
Diaryl azo derivatives as anti-diabetic and antimicrobial agents: synthesis, in vitro, kinetic and docking studies
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2021
Tehreem Tahir, Mirza Imran Shahzad, Rukhsana Tabassum, Muhammad Rafiq, Muhammad Ashfaq, Mubashir Hassan, Katarzyna Kotwica-Mojzych, Mariusz Mojzych
A series of kinetic assays was performed to determine the inhibition kinetics of the most active inhibitor from the series of tested compounds by following method32,33. The potential inhibitor with concentrations 0.00, 0.062, 0.125, 0.25, 0.5 mM was selected for kinetic studies. The substrate (pNPG) concentration was optimised from 5 to 40 mM in all kinetic studies. Pre-incubation and measurement time was the same as discussed in α-glucosidase inhibition assay. The assay was continuously monitored at 405 nm for 5 min at 30 s intervals in the microplate reader after the addition of the enzyme. The inhibition type on the enzyme was assayed by Lineweaver–Burk plots of the inverse of velocities (1/V) versus the inverse of substrate concentration 1/[S] mM−1, and the inhibition constant Ki was determined by two ways (Dixon plot of 1/V versus inhibitor concentrations) as well as secondary replot of slope versus inhibitor concentrations from Lineweaver–Burk plot.