Excess substrate inhibition

Excess substrate inhibited the oxidation of the p-hydroxyphenyl-pyruvate as did also benzoquinone acetic acid. This could be prevented by adding large amounts of ascorbic acid or small amounts of reduced 2,6-dichlorophenolindophenol. The latter was seven hundred times as effective as ascorbic acid with the purified enzyme 214), Zannoni and LaDu speculate that a product formed from p-hydroxyphenylpyruvate is the true inhibiting agent of the reaction. 

Any substance present in great excess can inhibit growth or even cause death. Metabolic products are often toxic to the organism that produces them. Thus, a batch fermentation can be limited by accumulation of products as well as by depletion of the substrate. A simple model for growth in the presence of an inhibitor is... 

Cell cultures can be inhibited by an excessive concentration of the substrate. One way to model substrate inhibition is to include an term in the denominator of the rate equation. See Equation (12.4). 

In our previous work [63], we studied the hydrolysis kinetics of lipase from Mucor javanicus in a modified Lewis cell (Fig. 4). Initial hydrolysis reaction rates (uri) were measured in the presence of lipase in the aqueous phase (borate buffer). Initial substrate (trilinolein) concentration (TLj) in the organic phase (octane) was between 0.05 and 8 mM. The presence of the interface with octane enhances hydrolysis [37]. Lineweaver-Burk plots of the kinetics curve (1/Uj.] = f( /TL)) gave straight lines, demonstrating that the hydrolysis reaction shows the expected kinetic behavior (Michaelis-Menten). Excess substrate results in reaction inhibition. Apparent parameters of the Michaelis equation were determined from the curve l/urj = f /TL) and substrate inhibition was determined from the curve 1/Uj.] =f(TL) ... 

Another type of inhibitor combines with the enzyme at a site which is often different from the substrate-binding site and as a result will inhibit the formation of the product by the breakdown of the normal enzyme-substrate complex. Such non-competitive inhibition is not reversed by the addition of excess substrate and generally the inhibitor shows no structural similarity to the substrate. Kinetic studies reveal a reduced value for the maximum activity of the enzyme but an unaltered value for the Michaelis constant (Figure 8.7). There are many examples of non-competitive inhibitors, many of which are regarded as poisons because of the crucial role of the inhibited enzyme. Cyanide ions, for instance, inhibit any enzyme in which either an iron or copper ion is part of the active site or prosthetic group, e.g. cytochrome c oxidase (EC 1.9.3.1). 

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 contrast to the AChR, AChE does not bind bungarotoxin or sulfhydryl reagents. It is inhibited by excess substrate (3 x I0 M), and the of electric eel AChE is about 10 M. The specific activity of the enzyme is one of the highest known 750 nmol/mg-hr, with a turnover time of 30-60 msec and a turnover number of 2-3 x 106. It is therefore one of the most efficient and fastest enzymes known. 

Acetylcholinesterase (AChE) In pesticides, an enzyme that will most rapidly hydrolyze acetylcholine as substrate, will not hydrolyze most non-choline esters, is inhibited by excess substrate, and is derived primarily from nervous tissue. 

Some substrates inhibit cell growth at high concentrations. Equation 4.9, one of the expressions for such cases, can be obtained on the assumption that excess substrate will inhibit cell growth, in analogy to the uncompetitive inhibition in enzyme reactions, for which Equation 3.42 holds ... 

The pentacyanocobaltate(II) ion has long been known to catalyze alkene hydrogenation, mainly of conjugated dienes. A review of the early work is available.45 The catalyst system shows negligible activity for the hydrogenation of non-activated monoenes. A major disadvantage is that the system is inhibited by excess substrate, and the turnover numbers obtained are generally less than 2. 

Glucuronidase from mammalian and non-mammalian sources, including the purified enzyme from female-rat preputial gland, often displays marked inhibition in the presence of excess substrate. The number of substrate molecules per active-enzyme center in the inactive enzyme-substrate complex ig24.100. ice.167 usuaUy 2, but values of166 3 and143 4 have also been reported. 

In Eq. (3) [S]o and [S]f are starting and ending substrate concentrations. S approaches [S] when substrate consumption is minimal, and S is substituted for [S] to correct for excess substrate consumption. In these analyses, however, substrate inhibition can be a problem if the product has a similar affinity to the substrate. Fortunately, most P450 oxidations produce products that are less hydrophobic than the substrates, resulting in lower affinities to the enzymes. There are exceptions, including desaturation reactions that produce alkenes from alkanes (10) and carbonyl compounds from alcohols. These products have hydrophobicities that are similar or increased relative to their substrates. 

Quenching Refers to the inactivation of a chemical activity by an excess of reactants or products. In enzymology, excess substrate or product may inhibit the enzymatic activity. 

The above process has been improved and optimized. An almost 400-fold increase in volumetric productivity relative to the published enzymic reaction conditions has been achieved, resulting in a attractive process that has been run on up to 100-g scale in a single batch at a rate of 30.6 g/L/hr. The catalyst load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These improvements were achieved by a combination of the discovery of a DERA with improved activity and reaction optimization to overcome substrate inhibition. The two stereogenic centers are set by DERA, with an ee of >99.9% and a diastereomeric excess of 96.6%. In addition, downstream chemical processes have been developed to convert the enzymic product efficiently to versatile intermediates applicable to the preparation of atorvastatin and rosuvastatin (Greenberg et al., 2004). 

Substrate preferences for AChEs and BuChEs vary with the species. Both mammal and bird AChEs rapidly hydrolyze ACh and its thiocholine analog acetylthiocholine (AcTh) (Silver 1974). Plasma-BuChE activity in the rat is reported to favor propionyl rather than butyryl substrates (Augustinsson 1948 Hoffmann et al. 1989). AChEs and BuChEs respond differently to increasing substrate concentration. AChEs are inhibited by excess substrate above 1-2 millimolars (mM) (Wilson et al. 1997 Silver 1974). BuChEs are less sensitive. BuChEs are preferentially inhibited by the selective inhibitor iso-OMPA and quinidine, and AChEs by the bisquatemary compound BW284c51. 

The effect of NMMA is attributable to its prevention of NO formation by NOS and its reversal by excess substrate (l-arginine) is a classic example of competitive enzyme inhibition (Figure 2). 

As well as alternative substrates, there have been a number of studies on inhibitors of flavocytochrome 62- Known inhibitors include D-lactate (16, 92-95), pyruvate (16, 58, 60, 96), propionate (96), DL-man-delate (90, 91), sulfite (60), and oxalate (16, 60, 97). Values of K, for these inhibitors and the conditions and types of enzyme used can be found in the papers referenced above. All of the above inhibitors show typical competitive inhibition except pyruvate and oxalate, for which mixed inhibition has been observed (60, 97). Inhibition has also been reported for excess substrate with the intact enzymes from both S. cerevisiae (16) and H. anomala (92), though not apparently with the cleaved enzyme from S. cerevisiae (16). It is possible that inhibition by excess substrate arises either from different binding modes at the active site or from a second lower affinity binding site elsewhere on the enzyme. 

Prefers ACh, is inhibited by excess substrates Found at neural junctions and in mammal RBCs and plasma and platelets of some vertebrates... 

Substrate inhibition is also observed for this enzyme system indeed, the kinetic lag is prolonged in the presence of excess substrate101). This is apparently due to the interaction of substrate with the yellow species resulting in the reduction of the yellow form to the native state concomittant with the formation of fatty acid radicals 93,102,103) jjPR studies showed that addition of substrate to the yellow form resulted in the disappearance of the observed EPR signals. Subsequent addition of hydroperoxide regenerated these signals93). 

Although it is quite simple to set up an experiment to determine the variation of v with [5], the exact value of V,n,y is not easily determined from hyperbolic curves. Furthermore, many enzymes deviate from ideal behavior at high substrate concentrations and indeed may be inhibited by excess substrate, so the calculated value of cannot be achieved in practice. In the past it was common practice to transform die Michaelis-Menten equation (9) into one of several reciprocal forms (equations [10] and [11]), and either 1/v was plotted against 1/[S], or [S]/v was plotted against [S]. 

When setting up methods of enzyme assay, it is necessary to (1) explore the relationship between reaction velocity and substrate concentration over a wide range, (2) determine K and (3) detect any inhibition at high substrate concentrations. Zero-order kinetics are maintained if the substrate is present in large excess (i.e., concentrations at least 10 and preferably 100 times that of the value of K ,). When [S] = 10 X K V is approximately 91% of the theoretical y,nax. The K , values for the majority of enzymes are of the order of 10 to 10" mol/L therefore substrate concentrations are usually chosen to be in tlie range of 0.001 to O.lOmol/L. On occasion, the optimal concentrations of substrate cannot be used (e.g., when the substrate has limited solubility or when the concentration of a given substrate inhibits the activity of another enzyme needed in a coupled reaction system). 

An excess of substrate inactivates the enzyme by forming compound III (red) or IV (emerald green). This substrate inhibition is very substantial in some of the current EIA procedures using POase (Tijssen et al., 1982). 

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