Inhibitor constant values

Fig. 4 presents the dependence of log k, and selectivity for NTE, log [ (NTE)/ i(AChE)], on the hydrophobicity (In. 7tCH2=0.5) of the R groups for the different compounds I-IV. The differential effect of changing the hydrophobicity on the anti-NTE and anti-AChE activities observed for four OP series suggests differences in the structure and inhibitory specificities of the active sites of the two target esterases [58,59], The calculated from the bimolecular inhibitor constants values of RIP = i(NTE)/ i(AChE) are presented in Table 1. 

Table 1. Additive hydrophobicity of R-groups and calculated from the bimolecu-lar inhibitor constants values of RIP = 1(NTE)/ i(AChE) for methyl (I) and phenylphosphonates (II) (la) and phosphates III, IV (lb). 

As discussed above, the degree of inhibition is indicated by the ratio of k3/k and defines an inhibitor constant (Kj) [Eq. (3.19)], whose value reports the dissociation of the enzyme-inhibitor complex (El) [Eq. (3.20)]. Deriving the equation for competitive inhibition under steady-state conditions leads to Eq. (3.21). Reciprocal plots of 1/v versus 1/5 (Lineweaver-Burk plots) as a function of various inhibitor concentrations readily reveal competitive inhibition and define their characteristic properties (Fig. 3.5). Notice that Vmax does not change. Irrespective of how much competitive inhibitor is present, its effect can be overcome by adding a sufficient amount of substrate, i.e., substrate can be added until Vmax is reached. Also notice that K i does change with inhibitor concentration therefore the Km that is measured in the presence of inhibitor is an apparent Km- The true KM can only be obtained in the absence of inhibitor. 

The buried Cys-212 of human carbonic anhydrase B (3 pM) is virtually unreactive towards 2-chloromercuric-4-nitrophenol (60 pM) at pH 9.2, but upon the addition of only 40 pM CN , the half-life drops to 10 minutes which is an, at least, 75-fold rate enhancement. On first analysis, this would suggest that inhibitor binding to the enzyme has produced a conformational change or altered the — SH environment of the Cys—212. This is unexpected. How would you prove by kinetic experiments that the CN is binding to the mercury compound and not the enzyme and that this is changing the reactivity. The rate reaches a constant value at high [CN ]. 

Acetylcholinesterase is subject to substrate Inhibition at high concentrations, but Mlchaells kinetics are observed at lower concentrations, because the substrate constant and the Mlchaells constant differ by a factor of 100. Turnover numbers run about 2-9 x 10 min l, and (Mlchaells constant) values are about 0.2 mM.76,116 Whatever the source, the enzyme is subject to inhibition by the same reversible and irreversible inhibitors. Most of the kinetic work has been done with the saline-extracted 11S enzyme from electric eel and the detergent-extracted 6S enzyme from erythrocytes. The former Is a tetramer derived from the native enzyme by the action of proteases the latter is a dimer. 

To determine inhibitor constants (K ), repeat step 17 in the presence of one or two different concentrations of inhibitor, [I]. Alternatively, for a Dixon Plot, test a range of inhibitor concentrations at two different substrate concentrations. Then plot 1/v against [I] for each value of [S]. 

Some representative examples are given of plant sources of the cited compounds (further plant sources can be readily accessed via the Web). ICjo (concentration for 50% inhibition) values are given in round brackets. Ka (dissociation constant) or K (enzyme-inhibitor dissociation constant) values are given in square brackets. For convenience compounds are grouped into alkaloids (also encompassing N-containing aromatic pseudoalkaloids), phenolics, terpenes and other compounds and are listed alphabetically within these four groupings. 

The electrostatic model for the micellar effect on the hydrolysis of phosphate monoesters is also consistent with the results of inhibition studies (Bunton et al., 1968, 1970). The CTAB catalyzed hydrolysis of the dinitrophenyl phosphate dianions was found to be inhibited by low concentrations of a number of salts (Fig. 9). Simple electrolytes such as sodium chloride, sodium phosphate, and disodium tetraborate had little effect on the micellar catalysis, but salts with bulky organic anions such as sodium p-toluenesulfonate and sodium salts of aryl carboxylic and phosphoric acids dramatically inhibited the micelle catalysis by CTAB. From equation 14 and Fig. 10, the inhibitor constants, K, were calculated (Bunton et al., 1968) and are given in Table 9. The linearity of the plots in Fig. 10 justifies the assumption that the inhibition is competitive and that incorporation of an inhibitor molecule in a micelle prevents incorporation of the substrate (see Section III). Comparison of the value of for phenyl phosphate and the values of K for 2,4-and 2,6-dinitrophenyl phosphates suggests that nitro groups assist the... 

In the case of compounds in Class 2, the elfect of the addition of increasing amounts of substances such as propene is important, for it is often found that although the addition of small amounts of propene results in a substantial decrease in the rate of decomposition of the compound, with further addition the rate attains a constant value which is unaltered by the presence of even very large amounts of propene i.e., a condition of maximal inhibition is attained. Inhibitors such as propene presumably act by replacing an active radical R with a relatively unre-active species, such as the resonance-stabilised allyl radical... 

Enzyme inhibition data are often presented as IC50, the concentration of the inhibitor to cause 50 percent inhibition at one chosen substrate concentration Kt, the inhibition constant (dissociation constant from the inhibitor-enzyme complex) determined by enzyme kinetic analysis (e.g., Dixon plot) and /Cin lcl, the time-dependent inhibition constant for mechanism-based inhibitors. IC50 values can be estimated from the study described earlier. A positive inhibition, defined as dose-dependent inhibition, with the inhibited activity lower than 50 percent of that of the negative control, will require further experimentation to define Ki for a better evaluation of in vivo inhibitory potential. Further, a study to determine Klwul may be performed to evaluate if the inhibitor acts via covalent binding to the active site of the enzyme, leading to time-dependent irreversible inhibition. 

Simultaneous to the graph creation, kinetic properties in each vRxn are used to create the appropriate reaction rate equations (ordinary differential equations, ODE). These properties include rate constants (e.g., Michaelis constant, Km, and maximum velocity, Vmax, for enzyme-catalyzed reactions, and k for nonenzymatic reactions), inhibitor constants, A) and modes of inhibition or allosterism. The total set of rate equations and specified initial conditions forms an initial value problem that is solved by a stiff ODE equation solver for the concentrations of all species as a function of time. The constituent transforms for the each virtual enzyme are compiled by carefully culling the literature for data on enzymes known to act on the chemicals and chemical metabolites of interest. 

This is the Michaelis-Menten equation, but with K , modified by a term including the inhibitor concentration and inhibitor constant. is unaltered. Therefore, curves of v against [5] in the presence and absence of inhibitor reach the same limiting value at high substrate concentrations, but when the inhibitor is present, JC, is apparently greater. Plots of 1/v against 1/[S] with and without inhibitor cut the ordinate at the same point but have different slopes and intercepts on the abscissa (Figure 8-9). 

Studied [87-90]. -Butanol (BU) is an inhibitor of the Zn(II)/Zn(Hg) electrode process, whereas TU accelerates this process [87]. With the increase of TU concentration, the standard rate constant values increased to the maximum value of O.lcms , which is reached at the concentration of 0.33 M TU. When BU was added to TU solutions, the rate of the Zn(II)/Zn(Hg) process decreased. For the molar concentration ratio [BU]/[TU] 12,... 

A low Km value reflects high affinity. At substrate concentrations S < < Km the reaction rate is directly proportional to the substrate concentration (first order reaction) at high substrate concentration (S >> Km) the reaction is zero order and is no longer dependent on the substrate concentration but only on the enzyme activity. To calculate Km and Umax as well as inhibitor constants it is advantageous to transform the Michaelis-Menten relation so as to obtain linear relationships between S and vq that can be evaluated graphically. An example is the Line-weaver-Burk equation, containing the reciprocal values of vo and S ... 

In the model presented in Eq. (8) a sound relationship, judged from the associated statistical parameters, is obtained between the PLS factors derived from MQSM and the inhibitor constant. These results are graphically represented in Fig. 3, where predicted vs. experimental values of log K are plotted. 

Although affinity constant values reported in the literature were measured under different experimental conditions of, e.g., pH, buffer type, and buffer concentration, several pH-dependent trends are apparent. According to such dependences, three classes of inhibitors can be identified" (Figure 2.15). In the... 

In competitive inhibition the inhibitor, I, binds reversibly to the active site of the enzyme. Consequently, the inhibitor competes with the substrate for the active site. As more substrate is added, at constant inhibitor concentration, the inhibitor is displaced and the reaction rate approaches the same maximum value as in the absence of inhibitor. However, more substrate is required to achieve any given reaction velocity in the presence of inhibitor than in its absence. The amount of inhibition depends on the inhibitor constant, Kl, which is also the dissociation constant for the binding of the inhibitor to the enzyme ... 

Further, continuous studies on S. pulmonaria by Tadesse and coworkers isolated two more new alkaloids pulmonarins A (19) and B (20) (Figure 2) [15]. The structures were determined as dibrominated quaternary ammonium salts by spectroscopic methods and confirmed by their synthesis as well. They found acetylcholinesterase (AChE) inhibitors and the most active inhibitor was pulmonarin B (20) with the inhibition constant value of 20 pM [15]. 

---