Product-inhibited enzymes

In this scheme, F symbolizes an essential metabolite, such as an amino acid or a nucleotide. In such systems, F, the essential end product, inhibits enzyme 1, xAie first step in the pathway. Therefore, when sufficient F is synthesized, it blocks further synthesis of itself. This phenomenon is called feedback inhibition or feedback regulation. 

Caramel color interacts with other food components. As an example, a concentration higher than 700 ppm caramel in cola increased the rate of hydrolysis of the aspartame, forming alpha-L-aspartyl-L-phenylalanine. Caramelization products inhibited enzymic browning by 85.8 and 72.2% when heated at pH 4 and 6, respectively, for 90 min. The highest inhibitory activity was found for the fraction with molecular weight of 1000 to 3000. Caramel is often used for adulteration of juices and other foods like honey or coffee. It can be determined by quantification of marker molecules such as 5-HMF, 4-Mel, and DFAs. ... 

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.

Product Inhibition, Enzyme Inactivation, and Substrate Recalcitrance... 

Figure 17-11 Two-step mechanism for phosphate ester hydrolysis. In the first step, substrate binds to the Fe ion, thereby displacing the bond from the -hydroxo ligand to this metal ion. The resulting Fe -coordinated hydroxide serves as the nucleophile in the hydrolysis reaction. The resulting species is a phosphate bridge, the same species observed in x-rays studies of product-inhibited enzyme.

Consequently, membrane bioreactors are an example of the combination of two unit operations in one step for example, membrane filtration with the chemical reaction. In a typical membrane bioreactor, as weU as acting as a support for the biocatalyst, the membrane can be a very effective separation system for undesirable reactions or products. The removal of a reaction product from the reaction environment can be easily achieved thanks to the membrane selective permeability, and this is of great advantage in thermodynamically unfavourable conditions, such as reversible reactions or product-inhibited enzyme reactions. A very interesting example of a membrane bioreactor is the combination of a membrane process, such as microfiltration or ultrafiltration (UF), with a suspended growth bioreactor. Such a set up is now widely used for municipal and industrial wastewater treatment, with some plants capable of treating waste from populations of up to 80 000 people (Judd, 2006). 

In the normal process ( ), step (J) occurs very rapidly and step (/) is the rate-determining step, whereas in the inhibition process (B), step (3) occurs very slowly, generally over a matter of days, so that it is rate determining. Thus it has been demonstrated with AChE that insecticides, eg, tetraethyl pyrophosphate and mevinphos, engage in first-order reactions with the enzyme the inhibited enzyme is a relatively stable phosphorylated compound containing one mole of phosphoms per mole of enzyme and as a result of the reaction, an equimolar quantity of alcohoHc or acidic product HX is hberated. 

FIG. 24-1 Feedback control. Product inhibits the first enzyme. 

Usually initial rates are measured in enzyme kinetics so as to avoid problems arising from kinetic complications such as product inhibition. 

Liver contains an enzyme called glucokinase, which also carries out the reaction in Figure 19.4 but is highly specific for D-glucose, has a much higher for glucose (approximately 10.0 mAf), and is not product-inhibited. With such... 

Substrate and product inhibitions analyses involved considerations of competitive, uncompetitive, non-competitive and mixed inhibition models. The kinetic studies of the enantiomeric hydrolysis reaction in the membrane reactor included inhibition effects by substrate (ibuprofen ester) and product (2-ethoxyethanol) while varying substrate concentration (5-50 mmol-I ). The initial reaction rate obtained from experimental data was used in the primary (Hanes-Woolf plot) and secondary plots (1/Vmax versus inhibitor concentration), which gave estimates of substrate inhibition (K[s) and product inhibition constants (A jp). The inhibitor constant (K[s or K[v) is a measure of enzyme-inhibitor affinity. It is the dissociation constant of the enzyme-inhibitor complex. 

The 2-ethoxyethanol was a by-product, as shown in Figure 5.13. The formation rate of 2-ethoxyethanol was the same as the conversion rate of the (S)- or (R)-ibuprofen ester one mole of 2-ethoxyethanol was formed when one mole of ester was catalysed. A known concentration of 2-ethoxyethanol was added in the organic phase before the start of the reaction for product inhibition. The plots of the kinetics for the free lipase system are presented in Figure 5.17 and immobilised enzyme (EMR) in Figure 5.18, respectively. The Kw value was 337.94 mmoFl 1 for the free lipase batch system and 354.20 mmoll 1 for immobilised... 

In non-competitive inhibition, the substrate (S) and inhibitor (I) have equal potential to bind to the free enzyme (E). The inhibitor forms a ternary complex with enzyme-substrate (ES) whereas the substrate will form another ternary complex with enzyme-inhibitor (El). Since the non-competitive inhibitor had no effect on the binding of substrate to the enzyme, the Km value remained consistent (or unchanged). There are two different ways for the formation of ESI ternary complex this complex would not form the product and therefore was decreased. Non-competitive inhibitor had no effect on substrate binding or the enzyme-substrate affinity, therefore the apparent rate constant (K ) was unchanged.5 A possible reason for product inhibition was because of the nature of 2-ethoxyethanol,... 

Fig. 5.19. Enzyme mechanism with non-competitive product inhibition.

However, there are disadvantages to using immobilised cells. The cell may contain numerous catalytically active enzymes, which may catalyse unwanted side reactions. Also, the cell membrane itself may serve as a diffusion barrier, and may reduce productivity. The matrix may sharply reduce productivity if the microorganism is sensitive to product inhibition. One of the disadvantages of immobilised cell reactors is that the physiological state of the microorganism cannot be controlled. 

Nitrilases catalyze the synthetically important hydrolysis of nitriles with formation of the corresponding carboxylic acids [4]. Scientists at Diversa expanded the collection of nitrilases by metagenome panning [56]. Nevertheless, in numerous cases the usual limitations of enzyme catalysis become visible, including poor or only moderate enantioselectivity, limited activity (substrate acceptance), and/or product inhibition. Diversa also reported the first example of the directed evolution of an enantioselective nitrilase [20]. An additional limitation had to be overcome, which is sometimes ignored, when enzymes are used as catalysts in synthetic organic chemistry product inhibition and/or decreased enantioselectivity at high substrate concentrations [20]. 

It is possible to deplete the brain of both DA and NA by inhibiting tyrosine hydroxylase but while NA may be reduced independently by inhibiting dopamine jS-hydroxylase, the enzyme that converts DA to NA, there is no way of specifically losing DA other than by destruction of its neurons (see below). In contrast, it is easier to augment DA than NA by giving the precursor dopa because of its rapid conversion to DA and the limit imposed on its further synthesis to NA by the restriction of dopamine S-hydroxylase to the vesicles of NA terminals. The activity of the rate-limiting enzyme tyrosine hydroxylase is controlled by the cytoplasmic concentration of DA (normal end-product inhibition), presynaptic dopamine autoreceptors (in addition to their effect on release) and impulse flow, which appears to increase the affinity of tyrosine hydroxylase for its tetrahydropteridine co-factor (see below). 

At first, it was thought that control of TH activity depended on inhibition by its end-product, noradrenaline, which competes with the binding of co-factor. According to this scheme, release of noradrenaline would diminish end-product inhibition of the enzyme and so ensure that synthesis is increased to replenish the stores. When the... 

However, as early as the 1970s, it was obvious that end-product inhibition of TH could not be the main factor regulating the rate of noradrenaline synthesis. Clearly, the hydroxylation of tyrosine takes place in the cytoplasm and so it must be cytoplasmic noradrenaline that governs enzyme activity. Yet, it is vesicle-bound transmitter that undergoes impulse-evoked release from the neuron. Also, when neurons are releasing noradrenaline, its reuptake from the synapse is increased and, even though some of this transmitter ends up in the vesicles, or is metabolised by MAO, there should be a transient increase in the concentration of cytoplasmic noradrenaline which would increase end-product inhibition of TH. 

Not a great deal is known about factors that actually activate tryptophan hydroxylase. In particular, the relative contribution of tryptophan supply versus factors that specifically modify enzyme activity under normal dietary conditions is unknown. However, removal of end-product inhibition of tryptophan hydroxylase has been firmly ruled out. Also, it has been established that this enzyme is activated by electrical stimulation of brain slices, even in the absence of any change in tryptophan concentration, and so other mechanisms are clearly involved. 

The degradation of pectin is initiated by the action of PME, an enzyme removing methylester groups from highly methoxylated pectins. Production of incompletely deesterified pectin, probably due to end-product inhibition of PME (19, 20), may explain that an affinity of PL for both pectate and pectin is required for full pathogenicity of the bacteria. 

The reaction rate for this enzyme kinetics example is expressed by the Michaelis-Menten equation and with product inhibition. 

Fig. 8.3 The proliferation curves of RNA strands (the Q beta system) for decreasing concentrations of added matrix molecules. If the number of matrix molecules is larger than that of the enzymes, a linear proliferation is observed (first curve). This slows down at high concentrations, due to product inhibition. RNA proliferation is exponential if the amount of enzyme is larger than that of the matrix. If no matrix is added, the system goes through an incubation phase and then forms an RNA sequence which is related to certain Q beta fragments (Eigen et al., 1982)...

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