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Disease Prediction and Drug Development
Published in Arvind Kumar Bansal, Javed Iqbal Khan, S. Kaisar Alam, Introduction to Computational Health Informatics, 2019
Arvind Kumar Bansal, Javed Iqbal Khan, S. Kaisar Alam
Michaelis–Menten equations (see Equations 10.5 and 10.6) are used to study the kinetics of enzyme's reaction. The rate of reaction is measured at different substrate concentration values. The Michaelis constant Km is the substrate concentration at which the reaction-rate is 50% of the maximum reaction-rate at the saturation point. The flux J through the linear pathway of n unimolecular reversible reaction is given by Michaelis–Menten equations. The substrates in the chain-reactions are denoted as Si (1 ≤ i ≤ n + 1) with S1 as the initial substrate and Sn+1 as the final substrate. The equilibrium constants K1→j is the product of reaction constants from reaction one to reaction Sj −1 → Sj. The coefficient is the Michaelis constant for the jth reaction.
J.B.S. Haldane (1892–1964)
Published in Krishna Dronamraju, A Century of Geneticists, 2018
Haldane’s biochemical work at Cambridge was noted for his contribution to enzyme kinetics. In 1925, G.E. Briggs and Haldane derived a new interpretation of the enzyme kinetics law described by Victor Henri in 1903, which was different from the 1913 Michaelis–Menten equation. Michaelis and Menten assumed that enzyme (catalyst) and substrate (reactant) are in equilibrium with their complex, which then dissociates to yield product and free enzyme. The derivation of the Briggs–Haldane equation is based on the quasi-steady-state approximation; that is, the concentration of intermediate complex (or complexes) does not change. As a result, the microscopic meaning of the “Michaelis Constant” (Km) is different. Most of the current models use the Briggs–Haldane derivation (Briggs and Haldane 1925).
Simple Enzyme Substrate Interactions
Published in John C. Matthews, Fundamentals of Receptor, Enzyme, and Transport Kinetics, 2017
What does the Michaelis constant tell us about the enzyme-substrate interaction? KM has the units of concentration, just like the KD. If we arbitrarily set [S] equal to KM, then Equation 162 becomes
In vitro assessment of the inhibitory effect of goreisan extract and its ingredients on the P-glycoprotein drug transporter and cytochrome P-450 metabolic enzymes
Published in Xenobiotica, 2022
Mikina Takiyama, Takashi Matsumoto, Noriko Kaifuchi, Yasuharu Mizuhara, Eiji Warabi, Katsuya Ohbuchi, Kazushige Mizoguchi
The metabolic activity of each sample (individual value) was calculated as follows: n = 2) and Ac is the metabolic activity of the vehicle control (mean value from n = 2). In analyses where the remaining activity in the presence of 10 µmol/L goreisan ingredients was 50.0% or less, the IC50 values were calculated using a nonlinear least squares method using pharmacokinetics analysis software (Phoenix WinNonlin 8.1) as follows: 0 was the inhibitory effect at C = 0. In the case of competitive inhibition of the CYP isoforms, the apparent Michaelis constant was calculated as follows:
Acoustic mist ionization mass spectrometry (AMI-MS) as a drug discovery platform
Published in Expert Opinion on Drug Discovery, 2019
Ian Sinclair, Gareth Davies, Hannah Semple
As with any biochemical assay, it is important to use a substrate concentration around Km, the Michaelis constant, which is the concentration at which 50% of enzyme is bound to substrate. This maximizes the diversity of modes of action identified during screening [28]. Determining Km by measuring initial rates of reaction at different substrate concentrations using AMI-MS can be carried out using two different methods, with either a live or stopped timecourse. A stopped assay, where the assay is run for a set length of time, followed by the reaction being terminated, allows the plates to be read multiple times if required, providing the product and substrate do not degrade over time. A live timecourse is an efficient method of measuring Km, where the assay is run at multiple substrate concentrations and read by AMI-MS, cycling through the plate multiple times (Figure 2). This is possible as each analysis only consumes ~10 nL of the 50 µL well volume, so measuring the same well-multiple times does not impact on the bulk volume of the well. If we were to explore 12 substrate concentrations, at n = 3, reading live it is only necessary to use 36 wells. Each of these wells can then be read every 2 min. If this same experiment was to be performed in a stopped timecourse around 500 wells would be needed; this greatly increases the volume of reagents required and increases the complexity of the experiment. In certain assays, a stopped timecourse may be a necessity if the stop reagent is required to enable the well to be ‘read’ by AMI-MS, as will be discussed in section 5.2.
In vitro evaluation of the inhibition and induction potential of olaparib, a potent poly(ADP-ribose) polymerase inhibitor, on cytochrome P450
Published in Xenobiotica, 2018
Alex McCormick, Helen Swaisland, Venkatesh Pilla Reddy, Maria Learoyd, Graeme Scarfe
Substrate concentrations selected were at approximately the Michaelis constant (Km) for each enzyme. For the determination of IC50 values, at least three replicate microsomal incubations were performed. The effects of furafylline, 8-methoxypsoralen, N,N′,N″-triethylenethiophosphoramide (thioTEPA), trimethoprim, sulfaphenazole, benzylnirvanol, quinidine, disulfiram and ketoconazole, known potent chemical inhibitors of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4/5, respectively, were also examined as positive control model inhibitors. The enzyme activities in these incubations were compared with vehicle samples containing an equivalent volume of methanol in place of olaparib solution.