Characterizing inhibition kinetics

Krungkrai, S. R., Aoki, S., Palacpac, N. M., Sato, D., Mitamura, T., Krungkrai, J., and Horii, T. (2004). Human malaria parasite orotate phosphoribosyltransferase Functional expression, characterization of kinetic reaction mechanism and inhibition profile. Mol. Biochem. Parasitol. 134, 245-255. 

Further characterization of the chemical outcome of the tritium label revealed that the tritium was only found in the reduced product dihydrofinasteride and not in finasteride. This suggested that finasteride binding was irreversible and all bound finasteride went through catalysis. The possibility that the product dihydrofi-nasteride was the actual inhibitor was ruled out when it was determined to be a simple, irreversible inhibitor of the enzyme with a A) = 1 nmol H. A more vexing problem for the researchers was that while the overall release of tritium from the H-fmasteride-enzyme complex was >98%, only 30 5% could be recovered as H-dihydrofmasteride. This, combined with the inhibition kinetics of dihydrofmasteride, suggested that the potent inhibitor compound had not yet been isolated. 

Inhibition of enzymes can basically be divided into reversible or irreversible. According to inhibition kinetics, it can be divided into three types— competitive, non competitive, and allosteric. Competitive inhibition can be characterized by binding of the inhibitor to the active site of the enzyme (they are structurally similar to substrate) and inhibition can be reversed by substrate access (reversible inhibition). The reaction rate is dependent on the substrate and inhibitor concentrations and their affinity to the enzyme. Noncompetitive inhibition cannot be reversed by substrate access and the inhibitor reacts with other parts of the enzyme rather than the active site, and it is not structurally similar to the substrate. The enzymatic reaction can be irreversible when the affinity of the inhibitor to the enzyme is relatively high. Allosteric ligands (inhibitors or activators) are bound to quite another... 

Because of the failure of the Monod equation to find universal applicability, many researchers have suggested variations on the form of this equation in attempts to better characterize the kinetic behavior of substrate-limited growth of microorganisms. There are several more complex mathematical models that take into account not only inhibition by substrates and/or products of biochemical reactions, but also other factors, such as cell death and cell maintenance effects and multiple limiting substrates (5,6). 

Zhang N, Seguin RP, Kunze KL, Zhang YY, Jeong H (2013) Characterization of inhibition kinetics of (S)-watfarin hydtoxylation by noscapine implications in warfarin therapy. Drug Metab Dispos 41 2114-2123... 

As with alternate substrate inhibitors, a progress curve or continuous enzyme assay is the most useful to begin to characterize the kinetics of inhibition. There can be immediate, or diffusion-limited inhibition of the... 

The Ca channels that have been the most extensively studied are the voltage-dependent Ca channels. These channels are usually found in plasma or transverse tubule membranes. Voltage-dependent Ca channels open in response to an appropriate membrane depolarization. Several different types of voltage-dependent Ca channels have been described and are characterized by differences in their activation and inactivation sensitivities to voltage, their kinetic properties, and their sensitivities to activation or inhibition by a variety of pharmacological agents. 

The carbon monoxide reaction is well studied and the observed kinetics are well understood. Of particular interest is the so-called CO-inhibiting regime , characterized by carbon dioxide covering and blocking the surface, so that the reaction rate is governed by CO desorption rate (see original citations in [78]). 

The effect of oxidizing atmospheres on the reduction of NO over rhodium surfaces has been investigated by kinetic and IR characterization studies with NO + CO + 02 mixtures on Rh(lll) [63], Similar kinetics was observed in the absence of oxygen in the gas phase, and the same adsorbed species were detected on the surface as well. This result contrasts with that from the molecular beam work [44], where 02 inhibits the reaction, perhaps because of the different relative adsorption probabilities of the three gas-phase species in the two types of experiments. On the other hand, it was also determined that the consumption of 02 is rate limited by the NO + CO adsorption-desorption... 

Aerobic The growth kinetics was described by an interacting, balanced and unstructured model characterized by phenol inhibition and oxygen limitation according to a double limiting kinetics [60, 62],... 

Depending on the oxidation conditions and its reactivity, the inhibitor InH and the formed radical In can participate in various reactions determining particular mechanisms of inhibited oxidation. Of the various mechanisms, one can distinguish 13 basic mechanisms, each of which is characterized by a minimal set of elementary steps and kinetic parameters [38,43 15], These mechanisms are described for the case of initiated chain oxidation when the initiation rate v = const, autoinitiation rate fc3[ROOH] -C vy and the concentration of dissolved dioxygen is sufficiently high for the efficient conversion of alkyl radicals into peroxyl radicals. The initiated oxidation of organic compounds includes the following steps (see Chapter 2). 

This problem was first approached in the work of Denisov [59] dealing with the autoxidation of hydrocarbon in the presence of an inhibitor, which was able to break chains in reactions with peroxyl radicals, while the radicals produced failed to contribute to chain propagation (see Chapter 5). The kinetics of inhibitor consumption and hydroperoxide accumulation were elucidated by a computer-aided numerical solution of a set of differential equations. In full agreement with the experiment, the induction period increased with the efficiency of the inhibitor characterized by the ratio of rate constants [59], An initiated inhibited reaction (vi = vi0 = const.) transforms into the autoinitiated chain reaction (vi = vio + k3[ROOH] > vi0) if the following condition is satisfied. 

In a study of the pectinesterase from bananas,64,85,102 three pectinesterase fractions were obtained after respective extraction with water, 150 mM sodium chloride, and 150 mM sodium chloride of pH 7.5. The fractions obtained were further purified by fractional salting-out with ammonium sulfate, and chromatography on columns of DEAE- and CM-cellulose. A 50-fold purification was achieved, and the individual, purified fractions were characterized with respect to different effects of cations, inhibition by sucrose, and reaction kinetics. 

Larsson, O. M., Falch, E., Krogsgaard-Larsen, P, and Schousboe, A. (1988) Kinetic characterization of inhibition of y-aminobutyric acid uptake into cultured neurons and astrocytes by 4,4-diphenyl-3-butenyl derivatives of nipecotic acid and guvacine. J. Neurochem. 50, 818-823. 

Thereafter, a reference text such as Enzyme Kinetics (Segel, 1993) should be consulted to determine whether or not the proposed mechanism has been described and characterized previously. For the example given, it would be found that the proposed mechanism corresponds to a system referred to as partial competitive inhibition, and an equation is provided which can be applied to the experimental data. If the data can be fitted successfully by applying the equation through nonlinear regression, the proposed mechanism would be supported further secondary graphing approaches to confirm the mechanism are also provided in texts such as Enzyme Kinetics, and values could be obtained for the various associated constants. If the data cannot be fitted successfully, the proposed reaction scheme should be revisited and altered appropriately, and the whole process repeated. 

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