Enzyme assay product inhibition

Figure E5.7 displays the kinetic progress curve of a typical enzyme-catalyzed reaction and illustrates the advantage of a kinetic assay. The rate of product formation decreases with time. This may be due to any combination of factors such as decrease in substrate concentration, denaturation of the enzyme, and product inhibition of the reaction. The solid line in Figure E5.7 represents the continuously measured time course of a reaction (kinetic assay). The true rate of the reaction is determined from the slope of the dashed line drawn tangent to the experimental result. From the data given, the rate is 5 jumoles of product formed per minute. Data from a fixed-time assay are also shown on Figure E5.7. If it is assumed that no product is present at the start of the reaction, then only a single measurement after a fixed period is necessary. This is shown by a circle on the experimental rate curve. The measured rate is now 16 jumoles of product formed every 5 minutes or about 3 /rmoles/minute, considerably lower than the rate derived from the continuous, kinetic assay. Which rate measurement is correct Obviously, the kinetic assay gives the true rate because it corrects for the decline in rate with time. The fixed-time assay can be improved by changing the time of the measurement, in this example, to 2 minutes of reaction time, when the experimental rate is still linear. It is possible to obtain...

Many drugs are effective as a result of inhibiting one or more enzymes. Enzyme inhibitors reduce the rate of formation of product. In a closed system such as an enzyme assay in a test tube, they do not alter the amount of product that is ultimately generated rather, it takes longer in the presence of an inhibitor to generate a given amount of product. 

Rate experiments that are typically carried out in the presence of different concentrations of an alternative product (or product analog) while using the normal substrates . This approach can be particularly useful when the normal product cannot be used because it is unstable, insoluble, or ineffective (the latter indicated by a very high Ki value). Moreover, the normal product may be consumed as an essential substrate in a coupled assay system for the primary enzyme. Fromm and Zewe used the alternative product inhibition approach in their study of hexokinase. Wratten and Cleland later applied this procedure to exclude the Theorell-Chance mechanism for liver alcohol dehydrogenase. See Abortive Complexes... 

The assay protocol should measure true initial rates (See Initial Rate Condition). For most systems, this represents a time period in which less than ten percent of the substrate concentration has undergone conversion. However, if a reaction is not significantly favored thermodynamically or if product inhibition is particularly potent, then a much smaller percentage of substrate conversion may be needed such that true initial rate conditions are obtained. Addition of an auxiliary enzyme system may prove necessary to avoid product accumulation. See Coupled Enzyme Assays... 

Figure Cl. 1.2 shows a typical time course resulting from a continuous assay of product formation in an enzyme-catalyzed reaction. The hyperbolic nature of the curve illustrates that the reaction rate decreases as the reaction nears completion. The reaction rate, at any given time, is the slope of the line tangent to the curve at the point corresponding to the time of interest. Reaction rates decrease as reactions progress for several reasons, including substrate depletion, reactant concentrations approaching equilibrium values (i.e., the reverse reaction becomes relevant), product inhibition, enzyme inactivation, and/or a change in reaction conditions (e.g., pH as the reaction proceeds). With respect to each of these reasons, their effects will be at a minimum in the initial phase of the reaction—i.e., under conditions corresponding to initial velocity measurements. Hence, the interpretation of initial velocity data is relatively simple and thus widely used in enzyme-related assays.

Enzyme kinetics are normally determined under steady-state, initial-rate conditions, which place several constraints on the incubation conditions. First, the amount of substrate should greatly exceed the enzyme concentration, and the consumption of substrate should be held to a minimum. Generally, the amount of substrate consumed should be held to less than 10%. This constraint ensures that accurate substrate concentration data are available for the kinetic analyses and minimizes the probability that product inhibition of the reaction will occur. This constraint can be problematic when the Km of the reaction is low, since the amount of product (10% of a low substrate concentration) may be below that needed for accurate product quantitation. One method to increase the substrate amount available is to use larger incubation volumes. For example, a 10-mL incubation has 10 times more substrate available than a 1-mL incubation. Another method is to increase the sensitivity of the assay, e.g., using mass spectral or radioisotope assays. When more than 10% of the substrate is consumed, the substrate concentration can be corrected via the integrated form of the rate equation (Dr. James Gillette, personal communication) ... 

The synthesized pyrroloquinazoline alkaloid as shown in Fig. 23 selectively inhibited COX-2 (IC5o = 1.2 pM) over COX-1 (IC50 > 10 pM) activity in human monocyte assays. In a purified ovine enzyme assay, the COX-1 activity was not affected even at a concentration over 50 pM compared to an IC50 value of 20.5 pM for COX-2 inhibition. The LOX efficacy was demonstrated by measuring LTB4 production in a mouse air pouch model [148]. 

Fig. 8. A quantitative enzyme assay of enzyme inhibition is shown. The ESI/MS analysis can be automated. Samples introduced include enzyme, substrate, product, potential inhibitor and internal standard. (A) Total ion current is measured for each sample. (B) Product ion formation is plotted with respect to internal standard. (C) If an inhibitor is found, the degree of inhibition is derived using ESI-MS...

Fluorogenic Substrates, Guilbault and Kramer (20) pubhshed a method using a fluorometric assay for anticholinesterase compoimds. The substrates used were nonfluorescent compounds, the acetyl and butyl esters of 1- and 2-naphthol, which are hydrolyzed by cholinesterase to highly fluorescent materials. The rate of change of fluorescence was related to enzyme activity, and inhibition was measured by decreased rate of change in the production of fluorescence. 

In t3 e 2 diabetes, hepatic glucose production is increased [113]. A possible way to suppress hepatic glucose production and lower blood glucose in type 2 diabetes patients may be through inhibition of hepatic glycogen phosphorylase [114]. In enzyme assay, Fosgerau et al. reported... 

Product inhibition is a cause of nonlinearity of reaction progress curves during fixed-time methods of enzyme assay. For example, oxaloacetate produced by the action of aspartate aminotransferase inhibits the enzyme, particularly the mitochondrial isoenzyme. The inhibitory product may be removed as it is formed by a coupled enzymatic reaction malate dehydrogenase converts the oxaloacetate to malate and at the same time oxidizes NADH to NADL... 

Fig. 4. ATPase activity of Hsp90. (a) Titration of ATPase activity in purified recombinant yeast ffsp90 by addition of geldanamycin. ATPase activity was measured using a continuous enzyme-coupled assay, as previously described (Panaretou et al, 1998), which avoids end-product inhibition by ADP. (b) Hsp90 ATPase activity is stimulated at higher temperatures to a greater degree than expected, suggesting some mechanism of activation by heat shock.

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