Product Inhibition of Enzymes

FIGURE 4.1 Block diagram of combined enzymatic hydrolysis and two-stage membrane separation (microfiltration (MF) and ultrafiltration) to remove sngars while retaining undigested biomass and enzymes. Reprinted from Andric et al. (2010b) with permission from Elsevier. 

There have been numerous empirical and semi-empirical models proposed for the enzymatic hydrolysis of cellulose that include inhibition terms (Andric et al., 2010a,b). The use of these models can be helpful with process design and economic calculations where it is necessary to account for the limitations imposed by inhibition. More rigorous mechanistic models for enzymatic hydrolysis have also included terms for product inhibition, as described in section 4.4. 

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SSF is a process in which the production of ethanol from cellulosic materials is achieved by utilizing cellulose, cellulase, ethanol-producing microbes and nutrients in the same reactor. This process is desirable because the continuous removal of sugars by fermentative organisms alleviates end-product inhibition of enzyme hydrolysis of cellulose. The process is also simplified because only one reactor is used. The SSF process for ethanol production from cellulosic materials was reported by Blotkamp et al. [65] and was later tested on a pilot scale (for detail, see [66]). 

The common aromatic pathway is subject to repression control of enzyme synthesis as well as the previously described control by end-product inhibition of enzyme activity. Much of the work is reviewed by Gibson and Pittard [3] and Doy [72]. Most of the studies have centered on repression control of DAMPS, the first enzyme in the pathway. The three DAMPS isoenzymes of E. coli are repressed by their specific aromatic amino acids—phenylalanine, tyrosine [107], and tryptophan [108]. In addition there is cross repression of DAMPS (tyr) synthesis by phenylalanine and tryptophan at high concentrations [107,109], and DAMPS (phe) synthesis is cross-repressed by tryptophan [109,110]. 

Cathepsin C is inhibited by excess substrate. Numerous examples of product inhibition of enzyme action are known. Phosphate inhibition of acid phosphomonoesterase activity is well documented (Chersi et al.. 

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. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

The two reactions are achieved in only one step without altering the product quality. The inhibition of enzymes by excess of intermediate components is reduced in this system. The presence of an organic solvent in the medium allows a high solubility of acylglycerols and a well-controlled partition of the components in the reactor. 

The ability of flavonoids (quercetin and rutin) to react with superoxide has been shown in both aqueous and aprotic media [59,94]. Then, the inhibitory activity of flavonoids in various enzymatic and nonenzymatic superoxide-producing systems has been studied. It was found that flavonoids may inhibit superoxide production by xanthine oxidase by both the scavenging of superoxide and the inhibition of enzyme activity, with the ratio of these two mechanisms depending on the structures of flavonoids (Table 29.4). As seen from Table 29.4, the data obtained by different authors may significantly differ. For example, in recent work [107] it was found that rutin was ineffective in the inhibition of xanthine oxidase that contradicts the previous results [108,109], The origins of such big differences are unknown. 

A substance that decreases the rate of an enzyme-catalysed reaction is known as an inhibitor and its effects may be permanent or transient. The inhibition of some reactions by substances which may be products of either that reaction or a subsequent reaction provides a control mechanism for cellular metabolism, while the selective inhibition of enzymes is the basis of many aspects of pharmacology and chemotherapy. 

Metabotropic receptors, in contrast, create their effects by activating an intracellular G protein. The metabotropic receptors are monomers with seven transmembrane domains. The activated G protein, in turn, may activate an ion channel from an intracellular site. Alternately, G proteins work by activation or inhibition of enzymes that produce intracellular messengers. For example, activation of adenylate cyclase increases production of cyclic adenosine monophosphate (cAMP). Other effector mechanisms include activation of phospholipases, diacylglycerol, creation of inositol phosphates, and production of arachidonic acid products. Ultimately, these cascades can result in protein phosphorylation. 

Figure 4.11 Effect of inhibition of enzyme 2 by cofactor A and of enzyme 1 by cofactor B (i.e., product inhibition) on the concentration of B in the basic system when operated as a fed-batch reactor. For the central and right panels the inhibition constants are indicated on top of each section. In the left panel, inhibition by products was not considered, and—indicates that the parameter is not applicable. Data presented in the left panel are taken from Figure 4.4. The values used for all other parameters ares given in Table 4.1, set I.

Herb-drug interactions are of growing concern due to the increased use and awareness of natural health products. They generally arise when natural health products inhibit CYP enzymes, altering the rate of metabolism for other drugs in the system. It is important to note, however, that interactions may also arise when... 

In summary, the combination of enzymes is advantageous from an enzymol-ogy and reachon engineering point of view. Reaction yields can be increased by avoiding product inhibition of single enzymatic reachons. Product decomposihon (e.g. by hydrolysis) can be overcome by further enzymatic transformahons. Tedious isolation of intermediate products is not necessary. However, both strategies - combinatorial biocatalysis and combinatorial biosynthesis - have their disadvantages. The in vitro approach needs every enzyme to be produced by recombinant techniques and purified in high amounts, which is in some cases difficult to achieve. On the other hand, product isolation from a biotransformation with permeabilized or whole host cells can be tedious and results in low yields. 

For the regulation of metabolic pathways metabolites are often used which are a product of that pathway. The basic strategy for the regulation is exemplified in the mechanisms employed in the biosynthetic and degradation pathways of amino acids, purines, pyrimidines, as well as in glycolysis. In most cases a metabolite (or similar molecule) of the pathway is utilized as the effector for the activation or inhibition of enzymes in that pathway. 

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