Inhibition by excess substrate

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. 

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. 

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. 

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... 

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]. 

Some control over the products can be gained by varying the cyanide-to-cobalt ratio (7). Other problems are that the catalyst is inhibited by excess substrate and that the lifetime of the catalyst is limited, especially if a stoichiometric amount of KCN is added. 

The animal diamine oxidase is inhibited by cyanide and other carbonyl reagents. In contrast to liver monoamine oxidase, diamine oxidase is not sensitive to p-chloromercuribenzoate or other SH reagents. Iso-nicotinoyl hydrazide is a potent inhibitor of diamine oxidase. The chemical basis for the inhibitions are not known. It has been suggested that the bacterial enzymes contain flavins, but there is no evidence for any cofactors in the animal enzymes. Again in contrast to monoamine oxidase, diamine oxidase is inhibited by excess substrate. This is interpreted as showing combination of the enzyme with separate substrate molecules at the two adsorbing sites in this situation there is no effective reaction, as the catalysis appears to require combination of the substrate at two points. 

A topical relation between the esteratic and anionic sites is implicit in any consideration of enzyme action in which the two sites participate. It seems reasonable to assume that the two sites are spaced to accommodate choline esters. The proximity of the sites is indicated by the ability of prostigmine to inhibit the reaction with thioacetic acid, which does not involve interaction with the anionic site. The necessity for binding acetylcholine at both sites for efficient catalysis has been used to explain inhibition by excess substrate. When high concentrations of acetylcholine are present, it is possible for one molecule to interact with the anionic site of the enzyme, while a second molecule associates with the... 

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.. 

Figure 2.23 graphically depicts a plot of I/vq versus l/[Aoj. The values V and Km are obtained from the intercepts of the ordinate (1/V) and of the abscissa (—1 /Km), respectively. If the data do not fit a straight line, then the system deviates from the required steady-state kinetics e. g., there is inhibition by excess substrate or the system is influenced by allosteric effects (cf. 2.5.1.3 allosteric enzymes do not obey Michaelis-Menten kinetics). 

From steady-state studies on the inhibition of xanthine oxidase by methanol at high and low xanthine concentrations, it has been possible to show that Mo does not participate in the direct binding of the first substrate molecule to form the enzyme-substrate complex ES. However, inhibition by excess substrate and formation of a complex ESS are due to the disruption of the electron-transfer chain at the stage where the molybdenum atom takes part in the transfer process. 

Random bi-substrate reactions can be distinguished from ordered reactions experimentally. The final reaction product can inhibit the overall reaction by competing with only the first (leading) substrate of the reaction. The reaction involving malate dehydrogenase outlined above is ordered and is inhibited by excess NADH, which competes with a normal leading-substrate NAD for binding to the enzyme. NADH does not, however, compete with the malate. 

Inhibition also occurs when products, substrates, or reagents acting as ligands stabilize the palladium intermediates too much, thus blocking the reaction sequence. Ligand excess may produce a similar effect. Thus many palladium-catalyzed reactions are inhibited by excess ligand whose electronic and steric properties must therefore be taken into account. 

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). 

During the following years, the picture became confused since acetylcholine-hydrolyzing enzymes obtained from various organs were found to have optimal activity at different concentrations of substrate. An explanation for this was put forward by Alles and Hawes (A13). They demonstrated the existence of two choline-esterases The first, whose activity was greatest at low concentrations and was inhibited by excess... 

Scott (S12) purified usual serum cholinesterase and cholinesterase from a homozygous silent individual having about 2 % of usual activity, which corresponds to silent type II cholinesterase. In most respects, the properties of the two purified cholinesterases were similar but not identical. The greatest differences noted were that the silent enzyme was more heat stable, and that, with it, there was less substrate inhibition by excess ben-zoylcholine. 

Activation at high substrate concentrations not only explains the failure of butyrylcholinesterase to follow simple Michaelis-Menton kinetics, but also explains the enigma of substrate inhibition of the enzyme using either benzoylcholine (A21, T7) or acetyl- or butyryl-salicylcholine as substrates. The proposal made by Hastings is analogous to that of Myers (M24, M25) for the inhibition of acetylcholinesterase by excess substrate, in this case acetylcholine. 

Acetylcholinesterase from human erythrocytes or rat brain homogenates reacts with acetylcholine in the presence of n-butanol in a manner similar to the benzoylcholine-horse enzyme system. The unusual behavior of the acetylcholine-horse enzyme system—in which inhibition of the enzyme by excess substrate does not occur (A21)—indicates that inhibition by n-butanol is probably competitive. Todrick et al. (T7) con-... 

The reaction rate will be slower owing to the removal of enzyme from the system. The El complex will be catalytically inert. The EIS complex may, however, be susceptible to reconversion to ES and make some contribution to catalytic activity. Noncompetitive inhibition cannot be reversed by excess substrate, but it may be reversed by exhaustive dialysis. 

Such inhibition involves covalent bonding at the active site and cannot be reversed by excess substrate or by dialysis. The site is therefore blocked and made catalytically inactive. Most inhibitors in this group are highly toxic, e.g. the organophosphorus nerve poisons. Thus, diisopropyl fluorophosphate (DFP) reacts irreversibly with the hydroxyl group of serine ... 

This reaction is characterized by a kinetics depending on three chemical parameters the uric acid concentration (which is the substrate and which will be denoted S), the oxygen concentration (which is the cosubstrate and which will be denoted A) and pH. To simplify the presentation, letters S and A will be used to denote the species themselves as well as their concentration. We shall only consider here the first two parameters S and A. pH is imposed much higher than the pK of uric acid in this way uric acid will always be considered as ionic. The kinetics corresponds to an inhibition by excess of substrate, and is of the first order for the cosubstrate the reaction rate is expressed as follows ... 

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). 

Investigations on phase ratio are also useful. This does not only affect partition and concentration of components (both water-soluble and poorly water-soluble), but also reduces the reaction inhibition by the product, the substrate excess or any other chemical inhibitor [37,40]. 

When the initial LA concentration is large, the quantity of substrate transferred to the aqueous phase allows the lipoxygenation to progress. This reaction consumes LA and produces HP, which favor the transfer of residual substrate between the two phases. Then catalysis and transfer have a reciprocal influence on each other. We demonstrated that the use of a non-allosteric enzyme in a compartmentalized medium permits the simulation of a co-operativity phenomenon. The optimal reaction rate in the two-phase system is reached for a high initial LA concentration 14 mM. Inhibition by substrate excess is observed in two-phase medium. 

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