Mercury-enzyme compounds

Papain. Papain is representative of a group of plant proteases. It is found in the latex of papaya as 6-7 per cent of the total protein. Enzymes of this group in general are activated by reducing agents, and the inclusion of cysteine in the extract stabilizes the enzyme and permits its purification to proceed smoothly. Papain has been crystallized from salt solutions and from 70 per cent ethanol. - Highly purified crystals of the inactive mercury salt have been obtained that are fully active on removal of the mercury. The mercury-enzyme compound has two enzyme molecules (M.W., 20,700) per mercury atom. 

The biochemical basis for the toxicity of mercury and mercury compounds results from its ability to form covalent bonds readily with sulfur. Prior to reaction with sulfur, however, the mercury must be metabolized to the divalent cation. When the sulfur is in the form of a sulfhydryl (— SH) group, divalent mercury replaces the hydrogen atom to form mercaptides, X—Hg— SR and Hg(SR)2, where X is an electronegative radical and R is protein (36). Sulfhydryl compounds are called mercaptans because of their ability to capture mercury. Even in low concentrations divalent mercury is capable of inactivating sulfhydryl enzymes and thus causes interference with cellular metaboHsm and function (31—34). Mercury also combines with other ligands of physiological importance such as phosphoryl, carboxyl, amide, and amine groups. It is unclear whether these latter interactions contribute to its toxicity (31,36). 

Mercuric chloride, other mercury-containing antibacterials and silver will inhibit enzymes in the membrane, and for that matter in the cytoplasm, which contain thiol, -SH, groups. A similar achon is shown by 2-bromo-2-nitropropan-l,3-diol (bronopol) and iso-thiazolones. Under appropriate condihons the toxic action on cell thiol groups may be reversed by addition of an extrinsic thiol compound, for example cysteine or thioglycollic aeid (see also Chapters 12 and 23). 

The degradation of phenylmercuric acetate to benzene, methylmercuric chloride to methane, and ethylmercuric chloride to ethane and Hg + is apparently carried out by different enzymes from the plasmid-carrying Escherichia coli strain K12 (R831) (Schottel 1978) and Pseudomonas sp. Resistance to organic mercury compounds has also been found in clinical isolates of nontuber-culous, rapidly growing mycobacteria (Steingrube et al. 1991) and can present a challenge in the clinical environment. 

Dimercaprol (British Anti-Lewisite or BAL) is a colorless, viscous oily compound with an offensive odor used in treating arsenic, mercury, and gold poisoning. It displaces the arsenic bound to enzymes. The enzymes are reactivated and can resume their normal biological activity. When given by injection, BAL can lead to alarming reactions that seem to pass in a few hours. 

The mercuric ion, Hg2 +, which is obtained after oxidation in the red blood cells and other tissues, is able to form many stable complexes with biologically important molecules or moieties such as sulphydryl groups. The affinity of mercury for sulphydryl groups is a major factor in the understanding of the biochemical properties of mercuric compounds, resulting in interference with membrane structure and function and with enzyme activity. 

Enzymes are important targets for mercury [71], and sulphydryl-group-containing enzyme being more sensitive to mercuric compounds than a non sulphydryl-group-containing enzyme [72], Enzymes reported to be inhibited include phosphatases [73, 74], dehydrogenases [75,76] and hexokinases [71]. 

The buried Cys-212 of human carbonic anhydrase B (3 pM) is virtually unreactive towards 2-chloromercuric-4-nitrophenol (60 pM) at pH 9.2, but upon the addition of only 40 pM CN , the half-life drops to 10 minutes which is an, at least, 75-fold rate enhancement. On first analysis, this would suggest that inhibitor binding to the enzyme has produced a conformational change or altered the — SH environment of the Cys—212. This is unexpected. How would you prove by kinetic experiments that the CN is binding to the mercury compound and not the enzyme and that this is changing the reactivity. The rate reaches a constant value at high [CN ]. 

There are several such toxic agents that cause considerable medical, public and political concern. Two examples are discussed here the heavy metal ions (e.g. lead, mercury, copper, cadmium) and the fluorophosphonates. Heavy metal ions readily form complexes with organic compounds which are lipid soluble so that they readily enter cells, where the ions bind to amino acid groups in the active site of enzymes. These two types of inhibitors are discussed in Boxes 3.5 and 3.6. There is also concern that some chemicals in the environment, (e.g. those found in industrial effluents, rubbish tips and agricultural sprays), although present at very low levels, can react with enhanced reactivity groups in enzymes. Consequently, only minute amounts concentrations are effective inhibitors and therefore can be toxic. It is suggested that they are responsible for some non-specific or even specific diseases (e.g. breast tumours). 

The treatment of metal poisoning is to administer a compound that binds the metal ion more strongly than does the group in the active centre of the enzyme. These compounds are known as chelating agents. For lead, the compound ethyl-enediaminetetraacetic acid (EDTA) is used. For mercury, dimercaptopropanol (dimercaprol) is used. 

The role of GSH in cellular protection (see below) means that if depleted of GSH, the cell is more vulnerable to toxic compounds. However, GSH is compartmentalized, and this compartmentalization exerts an influence on the relationship between GSH depletion or oxidation and injury. The loss of reduced GSH from the cell leaves other thiol groups, such as those in critical proteins, vulnerable to attack with subsequent oxidation, cross-linking, and formation of mixed disulfides or covalent adducts. The sulfydryl groups of proteins seem to be the most susceptible nucleophilic targets for attack, as shown by studies with paracetamol (see chap. 7), and are often crucial to the function of enzymes. Consequently, modification of thiol groups of enzyme proteins, such as by mercury and other heavy metals, often leads to inhibition of the enzyme function. Such enzymes may have critical endogenous roles such as the regulation of ion concentrations, active transport, or mitochondrial metabolism. There is... 

Once the fungicides penetrate to the cell membrane or into the cytoplasm they may operate by devious means to disrupt vital functions. There is substantial evidence that the quinones immobilize the sultliydryl and imino prosthetic group of enzymes. The 8-hydroxyquinoline and dithiocarbamate compounds are active against copper and other metallic members of an enzyme system, presumably hy their ability to chelate metals. Heavy metals such as mercury alTect certain enzymes such as amylases and may serve as general protein precipitants. 

The biochemical basis for the toxicity of mercury and mercury compounds resulls from its ability to form covalent bonds with sulfur. Even In low coiiccninilinns divalent mercury is capable of inaelivaiing enzymes containing suirhydrvl I —Nil) groups, causing iiileil crcncc with cellular metabolism and function. 

The behavior of aspartate carbamoylase changed dramatically when the enzyme was treated with the organic mercurial compound p-hydroxymercuribenzoate (see fig. 9.14). The binding of aspartate no longer showed positive cooperativity, and ATP or CTP were without effect. Exposure to mercurials was found to cause the enzyme to dissociate into two types of fragments, one of which retained the enzymatic activity but was no longer affected by CTP or... 

There are a few common and long-known uses of heavy metal compounds which should be mentioned. The barium meal is used in gastrointestinal X-rays. It is administered as barium sulfate, the high electron scattering of barium ions providing X-ray opacity. Mercurial diuretics, derivatives of propan-2-ol, work by inhibition of sulfhydryl enzyme sites. These compounds are now becoming obsolete, however. 

Nervous System. The nervous system is also a common target of toxic metals particularly, organic metal compounds (see Chapter 16). For example, methylmercury, because it is lipid soluble, readily crosses the blood-brain barrier and enters the nervous system. By contrast, inorganic mercury compounds, which are more water soluble, are less likely to enter the nervous system and are primarily nephrotoxicants. Likewise organic lead compounds are mainly neurotoxicants, whereas the first site of inorganic lead is enzyme inhibition (e.g., enzymes involved in heme synthesis). 

The lignin model compounds and their derivatives used in this study were custom synthesized at Queen s University by Dr. R. Bowers (Colour Your Enzyme). CIDEP and conventional ESR experiments were conducted using either a Varian E-104 spectrometer or a customized Bruker X-band spectrometer, modified similarly as previously described (7). The light source used for in situ irradiation was either a super high pressure 200 W mercury lamp, a Lambda-Physik EMG101-MSC XeCl excimer laser at 308 nm., or a Quanta-Ray GCR-11 Nd YAG solid state laser equipped for all four harmonic generations. 

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