Inhibition by high substrate concentration

The propionitrile nitrile hydratase-catalyzed reaction presented kinetics inhibited by high substrate concentrations that were clearly evidenced in the CSMR, as revealed in [33]. The kinetic parameters for nitrile hydratase-catalyzed reactions of... 

May be most inhibitors are mixed-type however competitive and non-competitive behaviors are frequently reported when one effect is significantly stronger than the other. Mixed-type inhibition, as non-competitive inhibition, can be partial or total depending on the activity of the tertiary enzyme-substrate-inhibitor complex. A particular case of mixed-type inhibition is uncompetitive inhibition in this case the enzyme has no preformed site for binding the inhibitor, that can only binds to the enzyme after the substrate has bound to it. This situation is not frequent, with the exception of the case when the substrate itself is the inhibitor in fact, uncompetitive inhibition by high substrate concentration is rather common in enzyme catalyzed reactions. 

Except for the case of uncompetitive inhibition by high substrate concentration (see Eq. 3.44), all mechanisms of inhibition can be represented by Eq. 3.45 (see, for instance, Eqs. 3.42 and 3.43) which is a very convenient expression for determining the kinetic parameters of the corresponding rate equations. 

A similar analysis can be made for any other type of inhibition. An interesting situation occurs in the case of uncompetitive inhibition by high substrate concentration. In this case, a steady-state analysis renders a third-order equation in ps that for certain values of the kinetic and diffusion parameters may give three positive values of Ps for one value of Po and a stability analysis should be made to assess the right value. The intermediate value is always unstable but the upper or lower... 

The presence of an organic phase in the bioreaction medium is only useful when the partition coefficient of at least one reactant is significantly greater than the unity. The characteristics of the selected solvent clearly influence the partition coefficients of substrates and products between the two phases. When enzyme is inhibited by high substrate or product concentrations, it is convenient to use solvents with high partition coefficients while the opposite has to be done when enzyme has low affinity with its substrate. Eggers et al. [29] define the overall biphasic concentration referred to the total volume of the system ... 

When acid DNase activity is assayed by the acid-solubility method the optimal DNA concentration is 0.4 mg/ml (9) and higher substrate concentrations appear to be inhibitory (16, 21, 34)- It has been shown, however, that this inhibition is because increasing substrate concentration decreases the efficiency of acid-soluble oligonucleotide release since the number of breaks per unit length of DNA is lower. If a direct method of estimating enzymic activity is used, such as the determination of phosphatase-sensitive phosphate, it can be shown that the inhibition by high substrate seen by the acid solubility method is only apparent (34)-... 

Inhibition Function. Incorporation of an inhibition function into the model in lieu of the Monod function is essential for process failure to be caused by high concentrations of volatile acids at residence times exceeding the wash-out residence time as is observed in the field. Koga and Humphry (IJ) have shown that continuous cultures obeying the Monod function are stable except at the wash-out residence time. The function proposed by Haldane (12) for the inhibition of enzymes by high substrate concentration will be used as an inhibition function and may be expressed as ... 

Inhibitors structurally related to the substrate may be bound to the enzyme active center and compete with the substrate (competitive inhibition). If the inhibitor is not only bound to the enzyme but also to the enzyme-substrate complex, the active center is usually deformed and its function is thus impaired in this case the substrate and the inhibitor do not compete with each other (noncompetitive inhibition). Competitive and noncompetitive inhibition effect the enzyme kinetics differently. A competitive inhibitor does not change but increases. Km (Fig. 25a) in contrast, noncompetitive inhibition results in an unchanged Km and an increased vmax (Fig. 25b). Some enzymes, e.g. invertase, are inhibited by high product concentration (product inhibition). 

Scheme 1.5, Fig. 1.3). This type of inhibition is relatively rare with single-substrate enzymes. It is not completely overcome by high substrate concentrations and lowers both and V by a factor of (1 + [I]/K ). No change occurs for k /K. ... 

The final conversion yield decreased when substrate concentration was increased from 2% to 4%. This was attributed to end product inhibition by the L-phenylalanine produced. Thus although faster conversion rates were observed with addition of high substrate concentrations, the product titres never exceeded 16 g l1. As already discussed the rate of yield of the conversion was proportional to the concentration of amino donor employed. Using a ratio of 1 3 substrate to amino donor, almost a 90% conversion was achieved in 3 hours. 

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]. 

The advantage of a two-phase lipoxygenation system lies in three points. They include avoidance of inhibition by the substrate as well as high solubility of the substrate in the organic phase and product recovery in the aqueous phase. Drouet et al. [36] improved the production yield of hydroperoxylinoleic acid at high concentrations of linoleic acid in highly stirred borate buffer-octane biphasic medium. 

For hydrogenation to take place, the substrate usually needs to bind to the metal complex, although exceptions are known to this rule [25]. Substrate inhibition can occur in a number of ways, for example if more than one molecule of substrate binds to the metal complex. At low concentration this may be a minor species, whereas at high substrate concentration this may be the only species. One example of this is the hydrogenation of allyl alcohol using Wilkinson s catalyst. Here, the rate dependence on the substrate concentration went through a maximum at 1.2 mmol IT1. The authors propose that this is caused by formation of a complex containing two molecules of allyl alcohol (Scheme 44.1) [26],... 

The micro-organisms involved in the AD process are highly sensitive to overloads and disturbances of several causes. For example, the methanogenic bacteria are inhibited by high concentrations of it own substrate (i.e., volatile fatty acids). This explains why AD processes can be easily destabilized. 

The reduction in enzymatic activity that results from the formation of nonproductive enzyme complexes at high substrate concentration. The most straightforward explanation for substrate inhibition is that a second set of lower affinity binding sites exists for a substrate, and occupancy of these sites ties up the enzyme in nonproductive or catalytically inefficient forms. Other explanations include (a) the removal of an essential active site metal ion or other cofactor from the enzyme by high concentrations of substrate, (b) an excess of unchelated substrate (such as ATP" , relative to the metal ion-substrate complex (such as CaATP or MgATP ) which is the true substrate and (c) the binding of a second molecule of substrate at a subsite of the normally occupied substrate binding pocket, such that neither substrate molecule can attain the catalytically active conformation". For multisubstrate enzymes, nonproductive dead-end complexes can also result in substrate inhibition in the presence of one of the reaction... 

In comparison with Equation 3.28 for the reaction without inhibition, the apparent value ofthe Michaelis constant increases by (K Ci)/Ky and hence the reaction rate decreases. At high substrate concentrations, the reaction rates approach the maximum value because large amounts of the substrate decrease the effect of the inhibitor. 

Noncompetitive inhibition cannot be completely reversed by very high substrate concentrations. Monod et al. defined for an allosteric enzyme a function of state R (Eq. 9-71) which is the fraction of total enzyme in the R (B) conformation ... 

In the presence of a large excess of Co2+, both native (97) and cobalt (92) carboxypeptidase A show an approximately two-fold activity increase. The kinetics of the enzyme are very complex at moderate or high substrate concentrations and involve both apparent activation and inhibition by substrate (95). Under the standard assay conditions used in connection with the observed cobalt activation, all these complicating factors contribute significantly. The additional Co2+ possibly interferes with these secondary effects rather than being a participant in catalysis. Further experimentation is needed to clarify this detail. 

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