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The Renin-Angiotensin System
Published in Austin E. Doyle, Frederick A. O. Mendelsohn, Trefor O. Morgan, Pharmacological and Therapeutic Aspects of Hypertension, 2020
There is some controversy as to the effect of circulating levels of plasma-renin substrate on its reaction rate with renin. A direct way of studying this problem is to measure both the kinetics of the reaction and circulating levels of substrate. This allows calculation of the effect of substrate concentration on reaction velocity using the Michaelis-Menten equation. However, the results reported using this approach in man have been very variable because of the wide range of values obtained for both circulating substrate and the marked discrepancies in the published values of the Km of the reaction, which vary over a 20- to 50-fold range.140,148,149,170,187,198,199 However, the results obtained for Km using purified substrate suggests that plasma-substrate levels are important determinants of reaction rates. This conclusion is supported by the fact that many groups have observed that the rate of formation of angiotensin I during in vitro incubation of human plasma (“PRA”) is increased in samples with elevated substrate concentrations.171,187,191,194,200
Enzyme Kinetics and Drugs as Enzyme Inhibitors
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
This Michaelis–Menten equation shows that vmax remains unaffected by a competitive inhibitor, but as competition means that more substrate is necessary in presence of such an inhibitor to reach the same level of ES as in absence of I, the apparent KM-value increases to KM ∙ (1 + (I)/KI). Thus, a plot of vi versus (S) reveals hyperbolas for different (I)-concentrations.
Pharmacokinetic-Pharmacodynamic Relationships of Cardiovascular Drugs
Published in Hartmut Derendorf, Günther Hochhaus, Handbook of Pharmacokinetic/Pharmacodynamic Correlation, 2019
Similar equations and models have been used to describe a variety of biochemical processes (e.g., protein binding, enzyme kinetics) since they all reflect reversible binding of a ligand to a receptor. The classic Michaelis-Menten equation, which relates the rate of a chemical reaction to the concentration of substrate, the maximum velocity (Vmax) and the Michaelis constant (Km), is in the form of Equation 7. The equation is also analogous to the Langmuir adsorption isotherm, which is used to describe the adsorption of gases to solid surfaces.5 Similarly, the association of oxygen and hemoglobin was described by Hill6 in 1910 using the equation:
Synthesis and biological activity evaluation of 3-(hetero) arylideneindolin-2-ones as potential c-Src inhibitors
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Salvatore Princiotto, Loana Musso, Fabrizio Manetti, Valentina Marcellini, Giovanni Maga, Emmanuele Crespan, Cecilia Perini, Nadia Zaffaroni, Giovanni Luca Beretta, Sabrina Dallavalle
N-Terminal His6-tagged Src was purchased from Merck-Millipore (cat. 14–326). The reaction was performed according to the manufacturer’s instructions with minor modifications: 500 μM Src-peptide (KVEKIGEGTYGVVYK), 100 μM ATP, 0.00087% NP-40, 10 ng of the enzyme, 10% DMSO in 10 μL at 30 °C for 10 min. To avoid peptide adsorption to the plastic surface, protein low-binding tubes were used. ADP-Glo Kinase Assay (Promega) was then used to detect kinase activity accordingly with manufacturer’s instruction with minor modifications. In details, reactions were transferred to white 384 well-plates and stopped by adding 10 µL of ADP-Glo Reagent (Promega) for 50 min at rt. 20 μL of Detection Reagent (Promega) was then added for 30 min and the reaction read using GloMax Discover microplate reader (Promega). Data were plotted using GraphPad Prism 5.0. ID50 values were obtained according to Equation (1)50 is the 50% inhibitory dose. Compounds tested were assumed to act as fully ATP-competitive inhibitors. Therefore, Ki values were calculated accordingly to Equation (2). Ki is the affinity of the inhibitor to the enzyme, [S] is the substrate (namely, ATP) concentration, and Km is the affinity of ATP calculated accordingly to Michaelis-Menten equation.
Enzyme-assisted modification of flavonoids from Matricaria chamomilla: antioxidant activity and inhibitory effect on digestive enzymes
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Elida Paula Dini de Franco, Fabiano Jares Contesini, Bianca Lima da Silva, Anna Maria Alves de Piloto Fernandes, Camila Wielewski Leme, João Pedro Gonçalves Cirino, Paula Renata Bueno Campos, Patrícia de Oliveira Carvalho
In order to measure the Michaelis–Menten constant, Km, the inhibition constant, Ki, and the Vmax (maximum reaction rate), a series of substrate concentrations (200–800 mM) was tested in the assay system. Each analysis was performed without (control) and with the chamomile infusion as inhibitor (40 and 60 µl). Vmax and Km were calculated and the type of inhibition kinetics was identified using the SigmaPlot software (Aspire Software International, Ashburn, VA). The Michaelis–Menten equation linearised by Lineweaver–Burk was used to determine Vmax and Km by plotting a graph, i.e. 1/V against 1/[substrate concentration], and estimated by the intercept and slope, respectively.
Evaluation of a flavonoids library for inhibition of pancreatic α-amylase towards a structure–activity relationship
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2019
Carina Proença, Marisa Freitas, Daniela Ribeiro, Sara M. Tomé, Eduardo F. T. Oliveira, Matilde F. Viegas, Alberto N. Araújo, Maria J. Ramos, Artur M. S. Silva, Pedro A. Fernandes, Eduarda Fernandes
It was described that the discrimination between mechanisms of enzymatic inhibition based on linear transformation of Michaelis–Menten equation is less accurate than the use of the nonlinear regression, since the transformation can also mask the variability inherent to kinetic data29,30. By the same reason, modelling on the means of repeated experiments instead of using individual data can also lead to misinterpreted mechanisms. In that sense, for the enzyme kinetic analysis, it was used the nonlinear regression to determine kinetic parameters and the type of inhibition of the most active flavonoids of each group. For this purpose, it was applied the Solver supplement of Microsoft Office Excel™. By means of iterative guesses for variables in each model equation, Solver was asked to minimize the sum of squared residuals between individual raw experimental results and the corresponding values generated by the model.