Metal-binding agent

There has been some uncertainty concerning the metal content of alkaline phosphatase and the role of zinc in the catalytic process. Early measurements by Plocke et al. (36, 50) showed that there were 2 g-atoms per dimer. The zinc requirement for enzymic activity was demonstrated by the inhibition of the enzyme with metal binding agents in accord with the order of the stability constants of their zinc complexes. It appears that in some cases (EDTA) zinc is removed from the enzyme and in other cases (CN) the ligand adds to the metalloprotein. A zinc-free inactive apoenzyme was formed by dialysis against 1,10-phenanthro-line. Complete activity was restored by zinc only zinc, cobalt, and possibly mercury produce active enzyme. 

Effects of inhibitors. All activities tested were inhibited by molybdate (33, 37), citrate (118), oxalate (118), and other metal-binding agents (119, 120), L-cysteine (48, 114, H9, 120), dithiothreitol (46, 114), and phlorizin (101). Where determined, Kt values calculated for the inhibition of the various activities by a given compound agreed closely (101, 118-120). 

Metal-binding agents (118-120) Oxalate, citrate, 1,10-phenanthroline, azide, diethyldithiocarbamate, cyanide, and 8-hydroxyquinoline inhibit, suggesting the metalloenzyme nature of the catalyst... 

Inhibition by a variety of metal-binding agents competitive with respect to phosphoryl substrates (118-120) has suggested that an enzyme-bound divalent cation (other than Mg2+) may participate also in the binding of phosphate substrates. Observed inhibition by p-chloro-mercuriphenyl sulfonate and iodoacetate suggests the possibility that sulfhydryl groups may also be involved at, or near, the active enzymic site (119, 120). 

The synthesis of crown ethers in 1967 fortunately occurred about the same time as naturally occurring lipophilic metal-binding agents were being identified as products of microbial growth. As the literature on synthetic and natural product ionophores is extensive274,275), there is no justification for a discussion on their chemistry here. 

Related Compounds sumably some antibiotics are delicately balanced so as to be able to compete successfully with the metal-binding agents of the bacteria while not disturbing the metal processing by the host. There is evidence that at least some bacteria have developed resistance to antibiotics through the development of altered enzyme systems that can compete successfully with the antibiotic.133 The action of the antibiotic need not be a simple competitive one. The chelating properties of the antibiotic may be used in metal transport across membranes or to attach the antibiotic to a specific site from which it can interfere with the growth of bacteria. 

The diflFerential reactivity of the zinc atoms in LADH has also been shown by a somewhat diflFerent type of experiment which is summarized in Table III. The native enzyme again contained a total of 3.5 gram atoms of zinc, of which only two are exposed and react with OP, as just shown. The remaining zinc is designated buried. When the enzyme is exposed to the metal-binding agent, sodium diethyldithiocarbamate. 

Few cases are known where a metal-binding agent is, of itself, highly injurious to an organism. Thus oxine 11.30) can enter the cells of bacteria and fungi without apparent injury (see Section 11.7.1). Such freedom from damage arises from the normal steric and affinity factors which allowed the active site to accumulate and retain the metal. 

The starting-point for these considerations must be the hard fact that the majority of chelating agents have no biological action. For example, very few of the metal-binding agents commonly used in analytical work are antibacterial (Albert, etal., 1947 Schraufstatter, 1950). 

Westerfeld, W.W. Effect of metal-binding agents on metalloproteins. Fed. Proc. 20, 158-176(1961)... 

Mahieu P, Buchet J-P, Lauwerys R (1987) Evolution clinique et biologique d une intoxication orale aigue par Tanhydride arsenieux et considerations sur I attitude therapeutique. J Toxicol Clin Exp 7 273-278 Maiorino RM, Aposhian HV (1985) Dimercaptan metal-binding agents influence the biotransformation of arsenite in the rabbit. Toxicol Appl Pharmacol 77 240-250 Marafante E, Vahter M (1984) The effect of methyltransferase inhibition on the metabolism of As]arsenite in mice and rabbits. Chem Biol Interact 50 49-57... 

A D-glutamic acid oxidase has been purified a himdredfold from Aaper-giUua ustus 103, 104). The enzyme deaminated n-glutamic acid and D-as-partic acid but had no activity with other DL-amino acids. The enzyme resembled that prepared from the octopus in its other properties 101). Thus, the optimum pH is 8 for both substrates and it appears, on the basis of inhibition and purification studies, that one enzyme is involved here as well. The enzyme is inhibited by 10 M concentration of KCN and p-chlo-romercuribenzoate but not by metal-binding agents. 

Whether the metal-binding agent is a polyelectrolyte or an ionic surfactant headgroup in a micelle, it is of interest to know the extent of binding of a metal ion to such a species/agent. Based on the work of Oosawa, Scamehorn et al. (1989) have suggested equations that relate the fraction of a metal ion that is bound to the micelle to the free (unbound) metal ions. The estimation of these fractions requires additional relations, such as the electroneutrality condition, concentrations of surfactants present as micelles... 

Metal-binding agents, including KCN, a,a -dipyridyl, o-phenanthro-line, and EDTA, did not show appreciable inhibition either by direct addition or by preincubation of the inhibitor with the enzyme (10 M, 2°C, 16 hours). The iron, therefore, must be bound extremely tightly to the protein in the case of pyrocatechase, but it can easily be dissociated and reconstructed in the case of some other phenolytic oxygenases (see Chapter 3). 

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