Metal-enzyme complexes

Metal-enzyme complexes, a subgroup of metal-protein complexes, exhibit enzymatic activity consequent to readily dissociable combination with a variety of metal ions. Many of these studies have been performed with unpurified enzymes, and, even when pure enzymes were used, the stoichiometry of the interaction of the metal and enzyme has not been measured. Enhancement of enzymatic activity as a result of the addition of metal ions and its partial loss on their removal has been the chief criterion of assessment of physiological significance. Only in a few instances, e.g., enolase, has the stability and stoichiometry been studied in relation to function (Malmstrom, 1953, 1954). The study of metal complexes and particularly metal chelates (Bjerrum, 1941 Martell and Calvin, 1952 Calvin, 1954) has provided both new experimental and new theoretical backgrounds for the study of metals in relation to the specificity of enzyme action, metal-enzyme (Calvin, 1954), metal-substrate (Najjar, 1951), and metalloenzyme interaction, as well as metal-enzyme inhibition (James, 1953). 

The above characterization of the metal-enzyme complexes is obviously not as satisfactory as that of the preceding group, being beset with many ambiguities and qualifications. Such limitations in our knowledge do not detract from the interest in these systems, but it is apparent that the establishment of more precisely defined chemical criteria is needed. 

The above categorization attempts to delineate metal-enzyme interactions in terms of structural and functional biochemistry and aims at the establishment of a working hypothesis and a subsequent operational approach. The characteristics of the metal-protein bond serve as the primary parameter for the differentiation of metalloenzymes from metal-enzyme complexes. The spectrum of bond strengths is continuous, of course, and the present discussion focuses attention on its extremes and not on its center, where overlapping behavior must be expected—a situation teleologically related to the behavior of acids and bases. 

Consideration of only the high bond-strength end of the metal-enzyme spectrum—the metalloenzymes—suggests a different investigative approach than is presently feasible with the metal-enzyme complexes. 

The additional comment that the high aflSnity of metalloenzymes for their metals as "compared with the stability of chelates which use the same ligands, argues against a thermodynamically strained coordination is similarly not relevant and based upon a misinterpretation of the entatic site hypothesis. Entasis implies that the difference in energy between the ground state and transition state for the enzymatic reaction is reduced, not that the metal-enzyme complex is thermodynamically less stable, as was inferred. Indeed, there is no reason to suppose that the distorted environment of a metal ion in an enzyme as opposed to a simple metal complex leads necessarily to an increase in free energy. The studies of alkaline phosphatase just presented certainly seem consistent with the entatic state hypothesis. 

Metal-enzyme complexes of iron, zinc, cobalt, and copper synthesis and characterization of model compounds for such enzymes X-ray single-crystal stmctural analysis of metaUoenzymes... 

The metal-ligand ratios and the formation constants of low M.W. complexes can be obtained by means of graphical methods such as Job s continuous variation [2], dilution method and molar ratios method [3,4], as well as by numerical methods [5] in the treatment of the complicated equilibria which have to be taken into account. For the polymeric complexes Job s method, which has been successfully applied to metal-enzyme complexes [6], Scatchard s method [7] and the numerical methods, modified for the polyelectrolyte effect, are still suitable. 

Trace metals including Mg, Zn, Cu, Mn, Fe, and Co are employed as cofactors in a number of enzymatic reactions. Indeed, for many enzymes the only cofactor is a metal ion. Two broad classes of metalutilizing enzymes are distinguishable by experimental means. These are the metalloenzymes where the metal is bound to the apoenzyme with sufficient strength to survive purification procedures and is present in a definite proportion to the protein, and the metal enzyme complexes where the metal, although easily dissociable, is required for full expression of enzyme activity. 

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

Lu W-P, PE Jablonski, M Rasche, JG Ferry, SW Ragsdale (1994) Characterization of the metal centers of the Ni/Fe-S component of the carbon-monoxide dehydrogenase enzyme complex of Methanosarcina ther-mophila. J Biol Chem 269 9736-9742. 

Elution of the bound antibody-enzyme conjugate occurs by only a slight shift in pH to acidic conditions or through the inclusion of a metal-chelating agent like EDTA or imidazole in the binding buffer. Either method of elution is mild compared to most immunoaffinity separation techniques (discussed in the previous section). Thus, purification of the antibody-enzyme complex can be done without damage to the activity of either component. 

Amino acid is one of the most important biological ligands. Researches on the coordination of metal-amino acid complexes will help us better understand the complicated behavior of the active site in a metal enzyme. Up to now many Ln-amino acid complexes [50] and 1 1 or 1 2 transition metal-amino acid complexes [51] with the structural motifs of mononuclear entity or chain have been synthesized. Recently, a series of polynuclear lanthanide clusters with amino acid as a ligand were reported (most of them display a Ln404-cubane structural motif) [52]. It is also well known that amino acids are useful ligands for the construction of polynuclear copper clusters [53-56], Several studies on polynuclear transition metal clusters with amino acids as ligands, such as [C03] [57,58], [Co2Pt2] [59], [Zn6] [60], and [Fe ] [61] were also reported. 

To successfully describe the structure and function of nitrogenase, it is important to understand the behavior of the metal-sulfur clusters that are a vital part of this complex enzyme. Metal-sulfur clusters are many, varied, and usually involved in redox processes carried out by the protein in which they constitute prosthetic centers. They may be characterized by the number of iron ions in the prosthetic center that is, rubredoxin (Rd) contains one Fe ion, ferredoxins (Fd) contain two or four Fe ions, and aconitase contains three Fe ions.7 In reference 18, Lippard and Berg present a more detailed description of iron-sulfur clusters only the [Fe4S4] cluster typical of that found in nitrogenase s Fe-protein is discussed in some detail here. The P-cluster and M center of MoFe-protein, which are more complex metal-sulfur complexes, are discussed in Sections 6.5.2. and 6.5.3. 

It should be added that MS-02 is not necessarily a mono-nuclear complex. It could be shown in a few cases that the catalytic activity of the metal ion is due to the formation of dinuclear metal-substrate complexes. Presumably in these species each oxygen atom of dioxygen coordinates to a different metal center. Such systems were extensively used to model the reactivity patterns of various enzymes containing a bimetallic active center. 

Probably the most effective use of XRF and TXRF continues to be in the analysis of samples of biological origin. For instance, TXRF has been used without a significant amount of sample preparation to determine the metal cofactors in enzyme complexes [86]. The protein content in a number of enzymes has been deduced through a TXRF of the sulfur content of the component methionine and cysteine [87]. It was found that for enzymes with low molecular weights and minor amounts of buffer components that a reliable determination of sulfur was possible. In other works, TXRF was used to determine trace elements in serum and homogenized brain samples [88], selenium and other trace elements in serum and urine [89], lead in whole human blood [90], and the Zn/Cu ratio in serum as a means to aid cancer diagnosis [91]. 

Most of the work on chiral recognition in the ground state deals with salts having chiral, primary alkylammonium cations. Another approach is the chiral discrimination between two enantiomeric anions present as counterions in metal-cation complexes (Lehn et al., 1978). Discrimination between enantiomeric transition states will be dealt with in the next section together with non-chiral mimicry of enzymic catalysis. 

Such metal-nitrosyl complexes have great physiological significance and are involved for example in enzyme regulation and protection against oxidation [62],... 

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