Inactivation of enzymes

Product Heat Treatment. Equivalent heat treatment for destmction of microorganisms or inactivation of enzymes can be represented by plotting the logarithm of time versus temperature. These relationships were originally developed for sterilization of food at 121.1°C, therefore the time to destroy the microorganism is the V value at 121.1°C (250°F). The slope of the curve is and the temperature span is one log cycle. The heat treatment at 131°C for one minute is equivalent to 121.1°C for 10 minutes (Fig. 10). 

Proteases (proteinases, peptidases, or proteolytic enzymes) are enzymes that break peptide bonds between amino acids of proteins. The process is called peptide cleavage, a common mechanism of activation or inactivation of enzymes. They use a molecule of water for this, and are thus classified as hydrolases. 

Pancreatic enzyme replacement is the mainstay of gastrointestinal therapy. Most enzyme products are formulated as capsules containing enteric-coated microspheres or microtablets to avoid inactivation of enzymes in the acidic stomach instead, they dissolve in the more alkaline environment of the duodenum. Capsules may be opened and the microbeads swallowed with food, as long as they are not chewed. A powder form is available for patients unable to swallow the capsules or microbeads, but bioavailability is poor. While products may contain similar enzyme ratios, they are not bioequivalent and cannot be substituted. Generic enzyme products generally display poor dissolution and should not be used.5 Table 13-3 lists commonly used enzyme replacement products. 

The inactivation of enzymes containing the zinc-thiolate moieties by peroxynitrite may initiate an important pathophysiological process. In 1995, Crow et al. [129] showed that peroxynitrite disrupts the zinc-thiolate center of yeast alcohol dehydrogenase with the rate constant of 3.9 + 1.3 x 1051 mol-1 s-1, yielding the zinc release and enzyme inactivation. Later on, it has been shown [130] that only one zinc atom from the two present in the alcohol dehydrogenase monomer is released in the reaction with peroxynitrite. Recently, Zou et al. [131] reported the same reaction of peroxynitrite with endothelial NO synthase, which is accompanied by the zinc release from the zinc-thiolate cluster and probably the formation of disulfide bonds between enzyme monomers. The destruction of zinc-thiolate cluster resulted in a decrease in NO synthesis and an increase in superoxide production. It has been proposed that such a process might be the mechanism of vascular disease development, which is enhanced by diabetes mellitus. 

In earlier studies the in vitro transition metal-catalyzed oxidation of proteins and the interaction of proteins with free radicals have been studied. In 1983, Levine [1] showed that the oxidative inactivation of enzymes and the oxidative modification of proteins resulted in the formation of protein carbonyl derivatives. These derivatives easily react with dinitrophenyl-hydrazine (DNPH) to form protein hydrazones, which were used for the detection of protein carbonyl content. Using this method and spin-trapping with PBN, it has been demonstrated [2,3] that protein oxidation and inactivation of glutamine synthetase (a key enzyme in the regulation of amino acid metabolism and the brain L-glutamate and y-aminobutyric acid levels) were sharply enhanced during ischemia- and reperfusion-induced injury in gerbil brain. 

The presence of lead and hexavalent chromium in these products is of chronic hazard concern and the EU has classified these pigments as harmful substances. Lead is soluble at stomach-acid concentrations and can accumulate in the organism. The results of a high lead intake include inactivation of enzymes and disturbances in the synthesis of haemoglobin. Hexavalent chromium compounds are considered to be carcinogenic. For these reasons the usage of lead chromate pigments has declined considerably in recent years. 

They also suggested the lipids were released and became extractable primarily through enzymatic reactions after maceration of the leaves for extraction. Thus, they postulated that the time interval between maceration of the plant tissue and the application of heat to coagulate proteins was critical. They recommended heat inactivation of enzymes and precipitation of proteins as quickly as possible after maceration to minimize this interference. 

Irreversible CYP inhibition can arise from different chemical mechanisms. However, a common initial step is the metabolic activation of a substrate into a reactive metabolite that is trapped within the active site of the CYP to form a tightly bound complex causing a long-lasting inactivation of enzyme activity. Enzymatic activity can be restored only through the new synthesis of the enzyme. For this reason, irreversible CYP inhibition is often referred to as mechanism-based inhibition , metabolite-based inhibition or suicide inhibition . 

In the earlier scheme, I represents a product formed by metabolism of the inhibitor by the enzyme. This product may be released into bulk solvent, or may interact (often covalently) with a suitably reactive component of the enzyme within the active site. This irreversibly inactivated enzyme complex is shown as El". There are two kinetic constants that can be obtained from relatively straightforward experiments with a suicide inhibitor. The Ki value is an equilibrium constant for the initial reversible step, and all the rate constants from the above scheme contribute to its value. The rate of irreversible inactivation of enzyme at a saturating concentration of the suicide inhibitor is given by fcinact. to which only k2> h, and k contribute (Silverman, 1995). At infinitely high concentrations of the inhibitor, the half-Ufe for inactivation is equal to ln2/ l inact ... 

The elimination or inactivation of enzymes used to treat proteins is a critical problem once the desired modification in functionality is achieved. In many instances, product inhibition or self destruction does not occur as noted above for fish protein concentrate. As stated by Puski (20), if heat inactivation is used, the proteins may be denatureT"and revert to insoluble forms. Washing out the enzyme at its isoelectric point would also remove a portion of the protein which is solubilized by the enzyme. Inactivation of enzymes by chemical means may also cause significant changes in the protein. Thus, while desired functional modifications of food ingredients may be obtained through enzyme treatment, the problem of latent enzyme activity in food formulations must be addressed. 

The heat sterilization of microorganisms and heat inactivation of enzymes are examples of first-order reactions. In the case of an enzyme being irreversibly heat-inactivated as follows ... 

The mechanism of hepatotoxicity is therefore currently unclear. It has been suggested that lipid peroxidation is responsible rather than covalent binding to protein. Arylation of other low molecular weight nucleophiles such as coenzyme A and pyridine nucleotides also occurs and may be involved in the toxicity. Bromobenzene is known to cause the inhibition or inactivation of enzymes containing SH groups. It also causes increased breakdown of phospholipids and inhibits enzymes involved in phospholipid synthesis. Arylation of sites on... 

Covalent modification of pyruvate kinase results in inactivation of enzyme. 

A. S. Ghatorae, G. Bell, and P. J. Halling, Inactivation of enzymes by organic solvents new technique with well-defined interfacial area, Biotechnol. Bioeng. 1994, 43, 331-336. 

T. J. Ahern and A. M. Klibanov, Analysis of processes causing thermal inactivation of enzymes, Methods Biochem. Anal. 1988, 33, 91-127. 

P. J. Halling, A. C. Ross, and G. Bell, Inactivation of enzymes at the aqueous-organic interface, Prog. Biotechnol. 1998, 15 (Stability and Stabilization of Biocatalysis), 365-372. 

They are believed to enhance the transbuccal permeation by a mechanism that is similar to that of bile salts, namely, extraction of lipids, protein denaturation, inactivation of enzymes, and swelling of tissues [39], Sodium dodecyl sulfate is reported to have a significant absorption enhancing effect but may also produce damage to the mucosa [13]. The effect of sodium... 

Transmission Electron Microscopy The primary site of surfactant activity appears to be the cell membrane (25, 32). Other effects have been reported such as the denaturation of proteins, inactivation of enzymes and inhibition of mitosis. 

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