Two types of enzyme inhibitors are of interest to food scientists: (1) low-affinity inhibitors and (2) high-affinity in hibitors. The former are effective in the millimolar to micromolar concentration range and readily dissociate from the enzyme; an example is inorganic phosphate inhibiting phytase. The latter are effective in the nanomolar to pi-comolar concentration range and are bound tightly to the enzyme; an example is soybean trypsin inhibitor. For low-affinity inhibitors the dissociation constant Ki is a useful parameter to know; for high-affinity inhibitors the amount present is usually of more concern.
Inhibition Model. A general equilibrium model of inhibition is shown in Figure 3. If the parameter a is very large, so that the species EIS does not exist, the inhibition is termed competitive (inhibitor competes with S for the enzyme). The effect, shown in Figure 4a, is that Vmax is unchanged, and Km increases. If a = 1 and /? = 0, EIS is formed but does not proceed to product P, noncompetitive inhibition results. As shown in Figure 4a, Vmax decreases and Km is unchanged. If a is greater than 1 but not extremely large, mixed inhibition occurs. These types are diagnosed by comparing the Hanes plots in the absence and presence of inhibitor (Figure 4b).
Inhibitor Constant. If the inhibition is competitive, KM is determined from the ratio of Kapp (the apparent value of K in the presence of inhibitor of concentration [/]) to KM (no inhibitor present): Kapp/KM = 1 + \J]IKL. If inhibition is noncompetitive, the ratio of true maximum velocity to apparent maximum velocity in the presence of inhibitor gives VmaxIVapp = 1 + [I\IKi. Determining Kn a, and fi in the cases of partial and mixed inhibition is too complex to be discussed here.
High-Affinity Inhibitors. Measuring the concentration of high-affinity inhibitors (eg, soy trypsin inhibitor) is relatively straightforward (13). Trypsin is mixed with aliquots of soy meal extract and after a brief incubation (for formation of the inhibitor-enzyme complex) the amount of uninhibited enzyme remaining is measured by a simple assay. The rate of reaction is plotted versus the size of the extract aliquot; the straight line through the data obtained at lower levels of inhibition intersects the x axis at a point where the amount of enzyme equals the amount of inhibitor present (Fig. 5) (14). In the example shown, 1.2 mL of soy meal extract contained a molar amount of trypsin inhibitor equal to the number of moles of trypsin used in each assay tube. If the absolute amount of trypsin is established by a titration assay, the amount of soy trypsin inhibitor can be expressed in absolute molar units rather than arbitrary trypsin inhibitor units (15). Conversely, if the absolute concentration of high-affinity inhibitor is known, this method serves to measure the molar amount of enzyme present.
Endogenous Inhibitors. Endogenous inhibitors may be present in crude extracts of materials containing the enzyme being assayed, resulting in a marked nonlinearity in the assay (Fig. 6a). The uninhibited rate may be found as follows (1). Let [e] be the amount of enzyme in the largest aliquot of extract used in making the plot of Figure 6. X is the fraction of that aliquot used for each of the other points.
Figure 2. Determining Vmax and Ku from experimental data using three different methods: (a) HYPER computer program: Vmax = 58.55, KM = 17.44, Std. Dev. 1.65; (b) Lineweaver-Burk plot: Vmax = 49.60, Km = 12.85, Std. Dev. 2.54; (c) Hanes plot: V^ = 58.96, KM = 17.56, Std. Dev. 1.66. Source: Data from Ref. 10.
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