Competitive inhibition conditions

Figure 4.8 Observed % inhibition as a function of [5]// for competitive (closed circles), noncompetitive (a = 1, closed squares) and uncompetitive (open circles) inhibition. Conditions used for simulation Ki = aKi = [/]. Note that the P>]/ATM axis is plotted on a logarithmic scale for clarity. 

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. 

U nder assay conditions whereby the concentration of the probe substrate is equivalent to Km, an IC50 estimate is equivalent to twofold K. For non-competitive inhibition, Kj is equivalent to IC50 since inhibitor and substrate binding are independent... 

Under conditions where the concentrations of the inhibitor and the substrate are much greater than that of the enzyme, the above equation holds. Therefore, for simple competitive inhibition, Kmts is unchanged, while the apparent value of K is given by ... 

Even this scheme represents a complex situation, for ES can be arrived at by alternative routes, making it impossible for an expression of the same form as the Michaelis-Menten equation to be derived using the general steady-state assumption. However, types of non-competitive inhibition consistent with the Michaelis-Menten type equation and a linear Linweaver-Burk plot can occur if the rapid-equilibrium assumption is valid (Appendix S.A3). In the simplest possible model, involving simple linear non-competitive inhibition, the substrate does not affect the inhibitor binding. Under these conditions, the reactions... 

Corey also pointed out that 16 reflects the transition-state of an enzyme-substrate complex. Its formation was later supported by the observation of Michaelis-Menten-type kinetics in dihydroxylation reactions and in competitive inhibition studies [37], This kinetic behavior was held responsible for the non-linearity in the Eyring diagrams, which would otherwise be inconsistent with a concerted mechanism. Contrary, Sharpless stated that the observed Michaelis-Menten behavior in the catalytic AD would result from a step other than osmylation. Kinetic studies on the stoichiometric AD of styrene under conditions that replicate the organic phase of the catalytic AD had revealed that the rate expression was clearly first-order in substrate over a wide range of concentrations [38],... 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

Characterization of a mechanism-based inhibitor may involve the estimation of the constants described in section IV, namely, /qnacI, Kh /ccat, and r. The most common approach has been to incubate inhibitor, enzyme, and cofactors together and to determine the decline in enzyme activity with time (26). In practice, this approach often employs the measurement of residual enzyme activity in a subsequent incubation with a specific substrate under conditions that limit further inactivation and competitive inhibition by the inactivator, usually by an appropriate dilution (10-fold or greater) of the original incubate (5). 

When the kinetics of the hydrolysis of rac-pantolactone by Lph were investigated a decrease in the reaction velocity was observed this was found to be because of competitive inhibition by D-pantolactone (Eq. 1) [19] and slight product inhibition of Lph. Under the same conditions of pH (7.5) and temperature (30 °C), l-pantolactone was completely converted to L-pantoic acid. This is certainly a disadvantage of Lph-catalyzed kinetic resolution, because space-time yields come to levels as low as 6 g L 1 h 1. 

Morris, E. R., Gidley, M. J., Murray, E.J., Powell, D. A., and Rees, D. A. (1980). Characterization of pectin gelation under conditions of low water activity, by circular dichroism, competitive inhibition and mechanical properties. Int. J. Biol. Macromol. 2 327-330. 

After Zemplen treatment under usual conditions, the sparingly water-soluble hexamers 49 and 51 were obtained in quantitative yields. Their modest solubility in water has however not jeopardized their biological evaluations in solid-phase competitive inhibition assays as well as in their cross-linking abilities with a model plant lectin, Concanavalin A, known to form cross-linked lattices in the presence of multiantennary glycans (39). 

Relates IC50 to Kt under conditions of competitive inhibition Kt equilibrium enzyme inhibitor dissociation constant Km Michaelis-Menton constant, [S] substrate concentration. 

Kinetic studies of the inhibitory effect of 1-butanol in different solvents on CALB have been performed and the competitive inhibition constant fQ values obtained correlated with the calculated activity coefficients of the substrate, suggesting that desolvation of the alcohol was the changing condition [35]. 

Spironolactone (Aldactone) is structurally similar to aldosterone and competitively inhibits its action in the distal tubule (exchange of potassium for sodium) excessive secretion of aldosterone contributes to fluid retention in hepatic cirrhosis, nephrotic syndrome and congestive cardiac failure (see specific use in chapter 24), in which conditions as well as in primary h)q)ersecretion (Conn s syndrome) spironolactone is most useful. Spironolactone is also useful in the treatment of resistant hypertension, where increased aldosterone sensitivity is increasingly recognised as a contributory factor. 

An enzyme catalyzing the reaction S P is competitively inhibited by I. The assay is based on the incorporation of label from radioactive S into P. = 2.3xio M and V max = 290 nmoles/min under standard assay conditions. -Ki = 2.3 X 10 M. The specific activity of the labeled S is 4.5 x lO CPM//i.mole. (a) What is the observed velocity in terms of CPM incorporated into P per minute when [S] = 2.3 X10 M (b) What would the observed rate be (CPM/min) if a fivefold excess of unlabeled S were added (c) What would the observed rate be (CPM/min) if a fivefold excess of I were added ... 

Probably, the intrinsic calcium is important for maintaining the protein in an active conformation (about 5 calcium atoms are present per molecule molecular weight, 750,000 calculated for 275 y of calcium/g. of enzyme), and extraneously added calcium is bound near the active site, thus blocking it, or reacts with one of the groups on the active site. The inhibition is instantaneous and of non-< ompetitive character, with an inhibitor constant (Ki) of 5.3 X 10 M at 20° and pH 7.6 (compare the non-competitive inhibition by beryllium with Ki = 2.9 X 10 Af under the same conditions). 

As well as alternative substrates, there have been a number of studies on inhibitors of flavocytochrome 62- Known inhibitors include D-lactate (16, 92-95), pyruvate (16, 58, 60, 96), propionate (96), DL-man-delate (90, 91), sulfite (60), and oxalate (16, 60, 97). Values of K, for these inhibitors and the conditions and types of enzyme used can be found in the papers referenced above. All of the above inhibitors show typical competitive inhibition except pyruvate and oxalate, for which mixed inhibition has been observed (60, 97). Inhibition has also been reported for excess substrate with the intact enzymes from both S. cerevisiae (16) and H. anomala (92), though not apparently with the cleaved enzyme from S. cerevisiae (16). It is possible that inhibition by excess substrate arises either from different binding modes at the active site or from a second lower affinity binding site elsewhere on the enzyme. 

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