Accumulation of intermediates

An important tool for elucidating the steps in the pathway was the use of metabolie inhibitors. Adding an enzyme inhibitor to a cell-free extract caused an accumulation of intermediates in the pathway prior to the point of inhibition (Figure 18.12). Each inhibitor was specific for a particular site in the sequence of metabolic events. As the arsenal of inhibitors was expanded, the individual steps in metabolism were revealed. 

Success with transgenic expression of Pds or Zds or both has not been reported. Also not known is twhether CRTISO and Z-ISO, the companion isomerases to the desaturations, will influence accumulation of intermediates or products. 

NAPLs can impede progress and growth of microorganisms Compound specific when chlorinated solvents are present Accumulation of intermediate compounds considered hazardous may result with chlorinated solvents... 

The reaction of ethyl 2,4,6-trinitrophenyl ether with aniline in dimethyl sulfoxide (DMSO) in the presence of Dabco occurs in two stages via the intermediate (6). Kinetic studies show that proton transfer is rate-limiting both in the formation of the intermediate and in the subsequent acid-catalysed decomposition to give 2,4,6-trinitrodiphenylamine. Phenoxide is a considerably better leaving group than ethoxide so that substitutions of phenyl 2,4,6-trinitrophenyl ethers and phenyl 2,4-dinitronaphthyl ether with aniline occur without the accumulation of intermediates. Both uncatalysed and base-catalysed pathways are involved. ... 

The above definition applies only if there is no accumulation of intermediates or formation of side products. lUPAC strongly suggests that this term be applied only when those conditions have been experimentally established. lUPAC also recommends that the terms rate of appearance or rate of disappearance (or rate of consumption ) be used if these conditions are not met. 

A X-ray crystallographic method for detecting the transient accumulation of intermediates in enzyme catalysis, protein folding, ligand-binding interactions, and other processes involving macromolecules. The approach is premised on the well documented retention of substantial reactivity of biological macromolecules, even in the crystalline state. 

The possible accumulation of intermediates during the photocatalytic oxidation of aromatic contaminants has been studied with a variety of techniques, including temperature-progranuned desorption (TPD), oxidation (TPO), and hydrogenation (TPH), Fourier transform infrared analysis (FTIR), and extraction of adsorbed species with a variety of solvents. 

Mendez-Roman and Cardona-Martinez [55] examined titanium dioxide catalysts with FTIR spectroscopy during the photocatalytic oxidation of toluene. Reaction intermediates, believed to be benzaldehyde and benzoic acid, were reported to accumulate on catalyst samples. This accumulation of intermediates was found to be reduced in the presence of gas-phase water. Mendez-Roman and Cardona-Martinez concluded that toluene appeared to be converted to benzaldehyde, which was then oxidized further to form benzoic acid. They suggested that the accumulation of benzoic acid led to the observed apparent catalyst deactivation. Other researchers, however, have argued that benzoic acid is unlikely to be the compound responsible for apparent deactivation in the photocatalytic oxidation of aromatics. For example, Larson and Falconer [43] concluded, based on higher CO2 evolution rates for benzoic acid relative to toluene during photooxidation, that benzoic acid was not sufficiently recalcitrant to be responsible for the deactivation seen with aromatic contaminants. 

The rates of the various reactions will vary. This may be due to the availability of cofactors, concentration of enzyme in a particular tissue, competition with other, possibly endogenous, substrates or to intrinsic factors within the enzymes involved. This variation in rates will clearly affect the concentrations of metabolites in tissues, and the half-life of parent compound and metabolites. It may lead to accumulation of intermediate metabolites. 

Some bacteria, including E. coli, have the full complement of enzymes for the glyoxylate and citric acid cycles in the cytosol and can therefore grow on acetate as their sole source of carbon and energy. The phosphoprotein phosphatase that activates isocitrate dehydrogenase is stimulated by intermediates of the citric acid cycle and glycolysis and by indicators of reduced cellular energy supply (Fig. 16-23). The same metabolites inhibit the protein kinase activity of the bifunctional polypeptide. Thus, the accumulation of intermediates of... 

Isolated from a strain of Pseudomonas, this enzyme contains bound FAD, which must be reduced by external NADH. Like a typical dioxygenase the enzyme introduces two atoms of oxygen into the product. However, it also uses the reduced FAD to reduce the double bond system (either before or after the attack by oxygen).381 Another enzyme of the same bacterium is remarkable in hydrolyzing the product of the oxygenation reaction to four different products without the accumulation of intermediates. 

The monomeric mechanism of chain assembly is characteristic for homopolysaccharides, which constitute the most difficult case for biosynthetic studies, as accumulation of intermediates could not be induced by removal of one of the glycosyl donors required for chain elongation. Among these polymers, the most extensive information has been obtained for polymers of JV-acetylneuraminic acid, namely, the capsular polysaccharides (36 and 37) of E. coli K1 (Ref. 354) and Neisseria meningitidis type c (Ref. 355), respectively. CMP-NeuAc serves as the glycosyl donor in the formation... 

Such burst experiments have since been performed on many other enzymes. But, bursts may be due to effects other than the accumulation of intermediates, and artifacts can occur. Some examples are the following ... 

E, Effects of rate optimization on accumulation of intermediates and internal equilibria in enzymes... 

The important conclusion is that specificity, in the sense of discrimination between two competing substrates, is determined by the ratios of kcJKM and not by Ku alone. Since km/KM is unaffected by nonproductive binding (section E) and by the accumulation of intermediates (section F), these phenomena do not affect specificity (see Chapter 13). Note that equation 3.44 holds at all concentrations of substrates. 

Where intermediates do accumulate, it is often through the use of synthetic, highly reactive substrates, such as esters with chymotrypsin, or through the effects of pH with alkaline phosphatase. There is a good theoretical explanation. It is a corollary of the principle of maximization of rate by the mutual increasing of kcat and Km that the accumulation of intermediates lowers the reaction rate.4 Any intermediate that does accumulate lowers the KM for the reaction, causing saturation at lower substrate concentrations. In other words, the accumulation of an intermediate means that the reaction has fallen into a thermodynamic pit. ... 

The decomposition of liquid water and the following reactions are the results of a typical chemical effect. In this case, however, overall water splitting does not occur because oxygen is not obtained but hydrogen and hydrogen peroxide are. On the other hand, it is impossible to decompose water by photochemical reaction under illumination with a xenon lamp. Although it is possible to decompose water by photocatalytic reaction using a desirable photocatalyst and photoirradiation, it is difficult to decompose in practice because of rapid backward reaction, the formation and accumulation of intermediates onto the surface of photocatalyst,10) and other reasons. 

Isomerization of 181 has been shown to lead to 2,5- and 2,4-dimethyl-phenols (189 and 190), respectively. At low acidity, 13% of 189 and 87% of 190 are formed. The rate of decomposition of arene oxide is the same as the rate of formation of these products. Under stronger acidic conditions the product ratio changes to 54% of 189 and 46% of 190. These discrepancies have been shown to arise from the accumulation of intermediate 182.91,92... 

When the reaction was carried out in the probe of a 31P NMR spectrometer, no evidence for the accumulation of intermediates was obtained (vide infra). 

M46. Murase, T., and Hakura, H., Accumulation of intermediate density lipoprotein in plasma after intravenous administration of hepatic triglyceride lipase antibody in rats. Atherosclerosis 38, 293-300 (1981). 

More recently, Bonfatti et al. (1999) have verified that the reactivity of glucose at Ti/Pt electrodes was acceptable in all current densities, slightly higher at 600A m-2 however, the electrochemical mineralization was low, particularly over a long electrolysis time, due to the accumulation of intermediates, mainly glucaric acid, which resisted further attack at the platinum electrode. The situation improved by increasing the temperature to 56°C. 

Fine-tuning of the enzymes in the biosynthetic pathway to prevent the accumulation of intermediates. 

Inhibitions of cell wall biosynthesis can be determined independently from DNA, RNA and protein biosyntheses. They will usually have been signaled by bactericidal effects and lysis of the test culture. In many instances, inhibition of cell wall synthesis results in the progressive accumulation of intermediates which can be assayed colorimetrically64, 6J) for N-acylaminohexose in acid-soluble fractions of the test bacteria. 

This feature implies that the phenol degradation kinetics did not change in the course of the runs due to the accumulation of intermediate products in the reaction ambient. As a consequence, only the phenol concentration values relative to conversion below 60% have been taken into account in the course of photoadsorption modeling. 

The available data suggest that in aqueous solution and at neutral pH prolyl isomerization proceeds according to a simple, one-step mechanism. Solvent water does not participate in the reaction and there is no accumulation of intermediates. The energy barrier to isomerization is enthalpic and represents the energy of resonance stabilization that is possessed by the C—N imide bond. 

In laying hens, induction of this riboflavin protein results in a 100-fold increase in plasma riboflavin, compared with males or nonlaying females. In mutant chickens lacking the protein, the adult has massive urinary loss of riboflavin. The embryo develops normally for about 10 days, then develops severe hypoglycemia associated with a reduction in medium-chain acyl coenzyme A (CoA) dehydrogenase to 20% of normal activity and the accumulation of intermediates of fatty acid oxidation (White, 1996). 

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