Metals, enzymatic binding

In the example described above, studies of a metal-substituted derivative helped in the evaluation of mechanistic possibilities for the enzymatic reaction. In addition, studies of such derivatives have provided useful information about the environment of the metal-ion binding sites. For example, metal-ion-substituted derivatives of CuZnSOD have been prepared with Cu , CuS Zn , Ag, Ni", or Co bound to the native copper site, and with Zn , Cu , Cu Co , Hg Cd, Ni", or Ag bound to the native zinc site. °° ° The SOD activities of these derivatives are interesting only those derivatives with copper in the copper site have a high degree of SOD activity, whereas the nature of the metal ion in the zinc site or even its absence has little or no effect. 

Type I IDI (IDI-I) are Zn -dependent metalloproteins which require a second divalent metal ion (Mg " ) for IPP binding via its diphosphate moiety (Fig. 87.6b, c) [94, 95]. The crystal structure of human IDI-I revealed a homodimeric stracture (Fig. 87.6a) with a six-coordinate metal ion binding pocket comprised of three histidine and two glutamate residues. These were initially suggested to bind Mn [96,97], but later on, the metal binding pocket was reassigned to have a much higher affinity for Zn ". Other divalent metal imis (Cd ", Co , Mg , Mn, Ni " ) are suitable substitutes and can, in some cases, even increase IDI-I activity [98, 99]. A participation of the metal catimi in the enzymatic reaction was delineated from X-ray analyses of enzyme-inhibitor complexes [94]. 

Phosphoamino acids that are part of proteins known to bind metal ions are posttranslational modifications introduced by specific protein kinases (Meggio et al, 1981 Vogel and Biidger, 1982c). The bovine milk protein casein and the hen egg-white protein ovalbumin, as well as possibly the human saliva acidic proline-iich proteins share sequence homology of their phosphorylated sites. Dephosphorylation of such sites by enzymatic phosphatase treatment usually reduces the affinity of such proteins for metal-ion binding (Bennick et al., 1981). Hence it is likely that dianionic phosphoryl moieties are directly involved in the complexation of metal ions. This seems particularly important for the two polyelectrolyte proteins that contain large amounts of phosphoserine residues, phosvitin purified from egg yolk (Ta-borsky, 1974), and the phosphoprotein purified from dentine (Linde et al, 1980). 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

Metallothioneins are a group of non-enzymatic, low-molecular mass (6-7 kDa) metal-binding proteins. They play an important role in the detoxification of a number (Zn, Cu, Cd, and Hg) of trace metals (Chassaigne and Lobinski 1998). 

Cyanide can inhibit enzymatic activity by binding to the metallic cofactor in metalloenzymes. 

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