Protein transition metal catalyzed reactions

Protein Modification Using Transition Metal Catalyzed Reactions... 

Divalent ions, such as the transition metals Fe, Co, Ni, Cu and Zn +, are most commonly used. The affinities of many retained proteins and their respective retention times, such as in the IDA chelator, are in the following order Cu > Ni > Zn > Co. The loss of metal ions at lower pH values leads to reduced adsorption capacity of the sorbent and can also cause damage to the target proteins by metal-catalyzed reactions. 

Apart from the applications in synthesizing drug molecules with a triazole linkage, azide-alkyne cycloaddition reactions have also been used for various biological applications such as site-specific protein/viruses modifications and functionalization of cell surfaces. Use of transition-metal-catalyzed reactions for... 

Transition metals such as iron can catalyze oxidation reactions in aqueous solution, which are known to cause modification of amino acid side chains and damage to polypeptide backbones (see Chapter 1, Section 1.1 Halliwell and Gutteridge, 1984 Kim et al., 1985 Tabor and Richardson, 1987). These reactions can oxidize thiols, create aldehydes and other carbonyls on certain amino acids, and even cleave peptide bonds. The purposeful use of metal-catalyzed oxidation in the study of protein interactions has been done to map interaction surfaces or identify which regions of biomolecules are in contact during specific affinity binding events. 

The NO/NO+ and NO/NO- self-exchange rates are quite slow (42). Therefore, the kinetics of nitric oxide electron transfer reactions are strongly affected by transition metal complexes, particularly by those that are labile and redox active which can serve to promote these reactions. Although iron is the most important metal target for nitric oxide in mammalian biology, other metal centers might also react with NO. For example, both cobalt (in the form of cobalamin) (43,44) and copper (in the form of different types of copper proteins) (45) have been identified as potential NO targets. In addition, a substantial fraction of the bacterial nitrite reductases (which catalyze reduction of NO2 to NO) are copper enzymes (46). The interactions of NO with such metal centers continue to be rich for further exploration. 

Some researchers have begun to explore the possibihty of combining transition metal catalysts with a protein to generate novel synthetic chemzymes . The transition metal can potentially provide access to novel reaction chemistry with the protein providing the asymmetric environment required for stereoselective transformations. In a recent example from Reetz s group, directed evolution techniques were used to improve the enantioselectivity of a biotinylated metal catalyst linked to streptavidin (Scheme 2.19). The Asn49Val mutant of streptavidin was shown to catalyze the enantioselective hydrogenation of a-acetamidoacrylic acid ester 46 with moderate enantiomeric excess [21]. 

Although hydroxyl radical is commonly assumed to be the most toxic of the oxygen radicals (with little direct evidence), other direct reactions are more likely to be important for understanding the cytotoxicity of peroxynitrite. A second oxidative pathway involves the heterolytic cleavage of peroxynitrite to form a nitronium-like species (N02 ), which is catalyzed hy transition metals (Beckman et al., 1992). Low molecular weight metal complexes as well as metals bound in superoxide dismutase and other proteins catalyze the nitration of a wide range of phenolics, including tyrosine residues in most proteins (Beckman et al., 1992). 

In general, transition metal ions are undesired in protein formulations because they can catalyze physical and chemical degradation reactions in proteins. However, specific metal ions are included in formulations when they are cofactors to proteins and in suspension formulations of proteins where they form coordination complexes (e.g., zinc suspension of insulin). Recently, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (63). 

One form of antioxidant defense may be the binding of excess Fe3+ and other transition metal ions, preventing Fe3+, and other transition metal pro-oxidants from catalyzing free radical reactions. Most intracellular Fe3+ is stored in ferritin. Mammalian ferritins consist of a hollow protein shell 12-13 nm outside diameter... 

Numerous transition metals ions form cluster complexes with chalcogenide anions [42-52], Iron and sulphur are unique elements in the sense that no two other elements can generate such a large diversity of cluster structures. This is the consequence of two stable oxidation states of iron ions and strong Fe-S bonds of significantly covalent character [53], Moreover, numerous structures are stable in several oxidation states, so these clusters serve as electron reservoirs in biological systems [51], This is why iron-sulphur proteins usually catalyze redox reactions. 

Protein phosphatases that are specific for phosphoserine/ phosphothreonine have a distinct reaction mechanism from tyrosine phosphatases. Protein serine phosphatases are transition metal-dependent, and the reaction mechanism does not involve a phosphoenzyme intermediate as in the case of PTPs. Crystal structures of multiple protein serine phosphatases have revealed how the enzymes catalyze hydrolysis of phosphoserine (14). 

Compounds such as superoxide anion and peroxides do not directly interact with lipids to initiate oxidation they interact with metals or oxygen to form reactive species. Superoxide anion is produced by the addition of an electron to the molecular oxygen. It participates in oxidative reactions because it can maintain transition metals in their active reduced state, can promote the release of metals that are bound to proteins, and can form the conjugated acid, perhydroxyl radical depending on pH, which is a catalyst of lipid oxidation (39). The enzyme superoxide dismu-tase that is found in tissues catalyzes the conversion of superoxide anion to hydrogen peroxide. 

The terminal oxidase in an energy-transducing, cytochrome-based electron-transport system maintains electron flow by coupling cytochrome oxidation to dioxygen (O2) reduction. Members of this protein class are referred to as cytochrome oxidases they carry out Oj-binding and redox chemistry at transition metal-containing active sites. Although iron is the most commonly used metal and may occur as a protoheme or iron-chlorin species in the protein, this section is concerned only with mitochondrial cytochrome oxidase, which contains 2 mol of Cn and 2 mol of heme a bound Fe per function unit. Biochemistry of the protein will not be considered here, instead the focus will be on the stmcture of the metal centers, on the reactions they catalyze and on models for these centers. 

Many of the active sites of today s proteins contain stably complexed transition metals (Mn, Fe, Co, Ni, Cu, Zn) thus providing these proteins and enzymes with catalytic properties which could not be achieved by other means. The choice of the metal depends on its ionic radius, on the reaction to be catalyzed, on the redox potential needed, on the type of ligands available and, last but not least, on the biological availability of the metal itself. 

Electron-transfer (ET) reactions play a central role in all biological systems ranging from energy conversion processes (e.g., photosynthesis and respiration) to the wide diversity of chemical transformations catalyzed by different enzymes (1). In the former, cascades of electron transport take place in the cells where multicentered macromolecules are found, often residing in membranes. The active centers of these proteins often contain transition metal ions [e.g., iron, molybdenum, manganese, and copper ions] or cofactors as nicotinamide adenine dinucleotide (NAD) and flavins. The question of evolutionary selection of specific structural elements in proteins performing ET processes is still a topic of considerable interest and discussion. Moreover, one key question is whether such stmctural elements are simply of physical nature (e.g., separation distance between redox partners) or of chemical nature (i.e., providing ET pathways that may enhance or reduce reaction rates). 

The metals are generally found either bound directly to proteins or in cofactors such as porphyrins or cobalamins, or in clusters that are in turn bound by the protein the ligands tire usually O, N, S, or C. Proteins with which transition metals and zinc are most commonly associated catalyze the intramolecular or intermolecular rearrangement of electrons. Although the redox properties of the metals are important in many of the reactions, in others the metal appears to contribute to the structure of the active state, e.g., zinc in the Cu-Zn dismutases and some of the iron in the photosynthetic reaction center. Sometimes equivalent reactions are catalyzed by proteins with different metal centers the metal binding sites and proteins have evolved separately for each type of metal center. 

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