Heavy metals enzyme inactivation

The enzyme is susceptible to heavy-metal inactivation, so that the biochemical manipulations were performed in the presence of (ethylenedinitrilo)tetraacetate (EDTA). The KM for D-arabinose 5-phos-... 

NADH as an end product. This implicates oxidized malic acid, either pyruvic or oxaloacetic acid, as another end product. By adding commercial preparations of L-lactic dehydrogenase or malic dehydrogenase to the reaction mixture, Morenzoni (90) concluded that the end product was pyruvic acid. Attempts were then made to show whether two enzymes—malate carboxy lyase and the classic malic enzyme, malate oxidoreductase (decarboxylating), were involved or if the two activities were on the same enzyme. The preponderance of evidence indicated that only one enzyme is involved. This evidence came from temperature inactivation studies, heavy-metal inhibition studies, and ratio measurements of the two activities of partially purified preparations of Schiitz and Radlers malo-lactic enzyme (76, 90). This is not the first case of a single enzyme having two different activities (91). 

Heavy metals such as Ag+, Hg2+, or Pb2+, which form complexes with the -SH groups, also inactivate the enzyme. For example, the poison phenylmercuric acetate is a potent inhibitor of urease. 

The stirring bar should be free of any slivers of magnetic material or heavy metals that could inactivate the enzyme. 

Inactivation can also be a consequence of oxidation of sulfhydryl groups, which can be avoided by the addition of mM concentrations of dithioerythritol or 2-mercap-toethanol to the buffer. Heavy metal ions can react with reactive groups on proteins, and Ca2+, Mg2+ and other divalent metal ions can promote the activity of degrading enzymes both of these effects can be suppressed by addition of mM concentrations ofEDTA. 

L-Arabinanases from A. niger have been shown19,55 to be inhibited or inactivated by certain heavy-metal ions, such as Hg2+ and Ag+. Fe3+ was also found to be slightly inhibitory (26%), whereas other metal cations (Mn2+, Fe2+, Cu2+, Zn2+, Ca2+, and Co2+) did not exert any significant effect on the activity. Urea (6 M) irreversibly denatured the A. niger enzyme.55... 

A pollutant may combine with the active site or sites of an enzyme thus inactivating it. For example, a heavy metal such as mercury, lead, or cadmium can attach itself to the thiol or sulfhydryl (SH) group on an enzyme molecule, forming a covalent bond with the sulfur atom. This will lead to inactivation of the enzyme if the sulfhydryl group... 

Fig. 2. MTs role in the subcellular distribution of heavy metals. Following uptake, metals activate MTF-1 to initiate transcription at MREs on MT promoters. Raised apo-thionein levels result in reduced toxicity of the heavy metal. Once the metals are bound to MT there is a decrease in free metals and a subsequent MTF-1 inactivation, and lowered MT gene transcription. When the rate of heavy metal accumulation exceeds that of MT synthesis, toxicity may occur. Binding of metals such as Cd and Hg may impair MTs role in regulating the levels of trace elements such as Zn and Cu, essential for maintaining enzyme activity. Other heavy metal binding proteins aid to protect from toxicity.

Glyceraldehyde-3-phosphate dehydrogenase, an enzyme in the glycolytic pathway (Chapter 8), is inactivated by alkylation with iodoacetate. Enzymes that use sulfhydryl groups to form covalent bonds with metal cofactors are often irreversibly inhibited by heavy metals (e.g., mercury and lead). The anemia in lead poisoning is caused in part because of lead binding to a sulfhydryl group of fer-rochelatase. Ferrochelatase catalyzes the insertion of Fe2+ into heme. 

Noncompetitive inhibition occurs when the inhibition depends only on the concentration of the inhibitor. This is usually caused by adsorption of the inhibitor at a site other than the active site but one which is necessary for activation. In other words, an inactive derivative of the enzyme is formed. Examples are the reaction of the heavy metals mercury, silver, and lead with sulfhydryl groups (—SH) on the enzyme. The sulfhydryl group is tied up by the heavy metal (ESH + Ag" " —> ESAg + H" ), and this reaction is irreversible. This is why heavy metals are poisons they inactivate enzymes in the body. 

Fourth, the presence of heavy metals cations, for example Cd(ll), Pb(ll) and Hg(ll) may lead to the inactivation of some enzymes (Vallee and Ulmer, 1972). The likelihood that an exchange of the bound metal between the labile complexes and the column increases... 

D. Heavy Metals Mercury and silver precipitate proteins and inactivate sulfhydryl groups of enzymes but are used rarely because of toxicity. Organic mercurials such as nitromersol and thimerosal frequently cause hypersensitivity reactions but continue to be used as preservatives for vaccines, antitoxins, and immune sera. Merbromin is a weak antiseptic and stains tissues a bright red color. In the past silver nitrate was commonly used for prevention of neonatal gonococcal ophthalmia, but it has been largely replaced by topical antibiotics. Silver sulfadiazine (a sulfonamide) is used to decrease bacterial colonization in bums. 

NeuSAc is a weak inhibitor of classical sialidases but does not act at all on the trans-sialidases. Correspondingly, the 2,3-difluoro derivative of NeuSAc, forming a covalent intermediate with sialidases and trans-sialidases (see later) was required at high concentrations (20 mM) to inactivate the enzyme completely Since trans-sialidases possess a lactose-binding site in their active center (as described next), lactose and its derivatives, especially lactitol, inhibit the sialic acid transfer of the enzyme from T. cruzi. Oligosaccharides from the mucins of this trypanosome can also inhibit the transfer of sialic acid to, for example, the substrate A-acetyllactosa-mine. It should be noted that heavy metal ions, especially merciuy, are potent inhibitors of sialidases and trans-sialidases. The latter enzymes do not require calcium ions, in contrast to some siaUdases. " " " ... 

One problem that may arise is when an antibiotic or preservative is the product, or part of the product under test. When this happens, the material must be inactivated or removed before sterility testing can take place. There are several methods of achieving this. Antibiotics, such as the penicillins, may be inactivated by the addition of the enzyme P-lactamase, whilst the action of sulphonamides can be blocked by the addition of />-aminobenzoic acid. Products containing preservatives or antimicrobial agents, such as benzoic acid, alcohols, or phenols, are diluted to the level at which the compound becomes ineffective. Products containing quaternary ammonium compounds can be inactivated by the addition of Tween, whilst many compounds containing heavy metals can be... 

The first major division of inhibitors is into two large groups of compounds irreversible and reversible inhibitors. Irreversible inhibitors are enzyme poisons, often chemically reactive compounds, which enter into chemical reactions with enz5nnes, forming irreversibly covalent bonds with the enzyme and reducing its activity to zero. An enzyme inactivated by an irreversible inhibitor caimot be reactivated by dialysis or a similar mild physical procedure. Many enzymes are poisoned by trace amounts of heavy metal ions this type of inhibition can be, in principle, reversed by dialysis against chelators such as EDTA or histidine. 

Heavy metals frequently act as enzyme inhibitors particularly when free -SH groups participate in the reaction. Papain, urease, myosin, triose phosphate dehydrogenase and many other enzymes fall into this category and are readily inactivated by Ca, Hg " " etc. The inhibition may sometimes be overcome by removing the metal ion, e.g. with H2S or with a chelating agent such as EDTA. 

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