Membrane enzymes thiol-containing

Copper is part of several essential enzymes including tyrosinase (melanin production), dopamine beta-hydroxylase (catecholamine production), copper-zinc superoxide dismutase (free radical detoxification), and cytochrome oxidase and ceruloplasmin (iron conversion) (Aaseth and Norseth 1986). All terrestrial animals contain copper as a constituent of cytochrome c oxidase, monophenol oxidase, plasma monoamine oxidase, and copper protein complexes (Schroeder et al. 1966). Excess copper causes a variety of toxic effects, including altered permeability of cellular membranes. The primary target for free cupric ions in the cellular membranes are thiol groups that reduce cupric (Cu+2) to cuprous (Cu+1) upon simultaneous oxidation to disulfides in the membrane. Cuprous ions are reoxidized to Cu+2 in the presence of molecular oxygen molecular oxygen is thereby converted to the toxic superoxide radical O2, which induces lipoperoxidation (Aaseth and Norseth 1986). 

Monoamine oxidase (MAO) (E.C. 1.4.3.4) is an enzyme found in all tissues and almost all cells, bound to the outer mitochondrial membrane. Its active site contains flavine adenine dinucleotide (FAD), which is bound to the cysteine of a -Ser-Gly-Gly-Cys-Tyr sequence. Ser and Tyr in this sequence suggest a nucleophilic environment, and histidine is necessary for the activity of the enzyme. Thiol reagents inhibit MAO. There are at least two classes of MAO binding sites, either on the same molecule or on different isozymes. They are designated as MAO-A, which is specific for 5-HT (serotonin) as a substrate, and MAO-B, which prefers phenylethylamine. Similarly, MAO inhibitors show a preference for one or the other active site, as discussed below. 

It was very early recognized that the calcium transport and the calcium-dependent ATPase could simultaneously be blocked by thiol reagents26). In contrast to various other thiol containing enzymes the activities of the sarcoplasmic reticulum membranes cannot be restored when the blocking agents are removed. 

S-Methylation is also an important pathway in the biotransformation of many sulfur-containing drugs. At least two separate enzymes, thiol methyltrans-ferase (TMT) and thiopurine methyltransferase (TPMT) are known to catalyze S-methylation in humans [87], TMT, a membrane-bound enzyme, catalyzes the S-methylation of captopril, D-penicillamine, and other aliphatic sulfhydryl compounds such as 2-mercaptoethanol. On the other hand TPMT, a cytosolic enzyme, catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds including 6-mercaptopurine and other thiopurines. Recently, S-methyltransferase has been shown to play a critical role in the metabolism of the antipsychotic drug, ziprasi-done, in humans [72, 88]. Both TPMT and TMT have been shown to be genetically polymorphic in humans. 

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). 

However, the reaction of NP with thiols may be a necessary but not sufficient cause for the release of NO from the ion as there are many thiols in frog heart tissue and NP is a vasodilator only under illumination. Furthermore Sogo et al. [50] could not detect NO generation from NP in human plasma containing cysteine, glutathione, homocysteine and reduced cysteine residues. Therefore, there must be a unique component of mammalian tissues which is involved in the release of NO from NP, and this reaction comes after reaction with thiol. Kowaluk et al. [51] report that NP is readily metabolised to NO in subcellular fractions of bovine coronary arterial smooth muscle and that the dominant site of metabolism is in the membrane fraction. This led to the isolation of a small membrane-bound protein or enzyme that can convert NP into NO. The mechanism shown in Scheme 8.2 combines the thiol reaction and that with an enzyme. 

It is generally accepted that the basis of the biological activity of mercury compounds is their reaction with the thiol groups, but the biological action is rather more complicated. Frank (1955) showed that mercury compounds can influence the effect of enzymes which do not contain thiol groups. Mercury also reacts with the phosphoryl groups of the cell membranes (Bassow et a/., 1961) and with the amino and carboxyl groups of the enzymes (Lipscomb et al., 1968). Webb (1966) lists more than 40 enzymes inhibited by mercury compounds. 

Inorganic and organic mercury compounds have a strong affinity for thiol chemical groups. Most proteins and aU enzymes contain these groups so that mercury readily is bound to body tissues. Most mercury compounds are potent enzyme inhibitors which affects membrane permeability, which in turn affects nerve conduction and tissue respiration. 

---