Reductases ribonucleotide

Ribonucleotide reductase, an allosteric enzyme from Escherichia coli, converts ribonucleotide diphosphates to the corresponding deoxyribo-nucleotides, and therefore provides the necessary precursors for DNA synthesis (Reichard, 1967). During purification ribonucleotide reductase separates into two nonidentical subunits, proteins B1 and B2, each enzymatically inactive (Brown et a/., 1969a). The active enzyme is formed in the presence of magnesium ions and consists of a 1 1 complex of the two subunits (Thelander, 1973). Proton subunit B2 (mol. wt. 78,000), which participates in the formation of the catalytic site (Thelander et al, 1976), contains nonheme iron (Brown et al, 1969b) and an ESR-detectable organic 

FIGURE 6. The role of free radicals in the reduction of ribonucleotides to 2 -deoxyribonu-cleotides catalyzed by AdoCbl-dependent ribonucleotide triphosphate reductase. 

The RNRs are divided into four classes, depending on the cofactors utilized to catalyze the reaction [45]. Class I RNR, which is found in e.g. mammals and E.coli bacteria, employs a stable neutral tyrosyl radical coupled to a di-iron (Fe202) cluster [46]. Class II uses 5 -deoxy-5 -adenozyl-cobalamin [47] (the active form of vitamin - see also the chapter by Smith, Wetmore and 

Radom). Class IE is also found in E. coli, when grown under anaerobic conditions, and uses a neutral glycyl radical as cofactor, similarly to the previously described anaerobic PFL enzyme [48]. Class IV, finally contains what is again beheved to be a tyrosyl radical, this time linked to a di-manganese cluster [49]. In addition, class 1 RNRs have been divided into subclasses la and lb, differing in e.g. their expression mechanism [50]. We will in this chapter consider aerobic Class I RNRs only. 

In order to understand the radical transfer mechanism between Tyrl22 and the active site, we first need to consider the protonation state of the tyrosyl radical. In Table 4 we list the EPR parameters (a-protons) and unpaired spin density distributions of neutral vs charged tyrosyl radicals, and compare the data with results obtained for Y122 in wild type E. coli RNR. From the data listed, it is clear that the tyrosyl radical is neutral, which has important implications for the radical transfer - i.e., that this is not a case of pure electron transfer but 

The final step of the radical transfer is H-atom migration between tyrosyl radical Y730 and C439. This has a barrier of ca 8 kcal/mol, and is only slightly endothermic (0.4 kcal/mol). The overall radical transfer between Y122(R2) and 

We have in the present chapter shown results from theoretical model system studies of the catalytic reaction mechanisms of three radical enzymes Galatose oxidase. Pyruvate formate-lyase and Ribonucleotide reductase. It is concluded that small models of the key parts of the active sites in combination with the DPT hybrid functional B3LYP and large basis sets provides a good description of the catalytic machineries, with low barriers for the rate determining steps and moderate overall exothermicity. The models employed are furthermore able to reproduce all the observed features in terms of spin distributions and reactive intermediates. 

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Figure 1.9 Examples of functionally important intrinsic metal atoms in proteins, (a) The di-iron center of the enzyme ribonucleotide reductase. Two iron atoms form a redox center that produces a free radical in a nearby tyrosine side chain. The iron atoms are bridged by a glutamic acid residue and a negatively charged oxygen atom called a p-oxo bridge. The coordination of the iron atoms is completed by histidine, aspartic acid, and glutamic acid side chains as well as water molecules, (b) The catalytically active zinc atom in the enzyme alcohol dehydrogenase. The zinc atom is coordinated to the protein by one histidine and two cysteine side chains. During catalysis zinc binds an alcohol molecule in a suitable position for hydride transfer to the coenzyme moiety, a nicotinamide, [(a) Adapted from P. Nordlund et al., Nature 345 593-598, 1990.)...

Nordlund, P., Sjoberg, B.-M., Eklund, H. Three-dimensional stmcture of the free radical protein of ribonucleotide reductase. Nature 345 593-598, 1990. 

Uhlin, U., Eklund, H. Structure of ribonucleotide reductase protein Rl. Nature 370 553-559, 1994. 

The first two of these are mediated by 5 -deoxyadenosylcobalamin, whereas methyl transfers are effected by methylcobalamin. The mechanism of ribonucleotide reductase is discussed in Chapter 27. Methyl group transfers that employ tetrahydrofolate as a coenzyme are described later in this chapter. 

When induced in macrophages, iNOS produces large amounts of NO which represents a major cytotoxic principle of those cells. Due to its affinity to protein-bound iron, NO can inhibit a number of key enzymes that contain iron in their catalytic centers. These include ribonucleotide reductase (rate-limiting in DNA replication), iron-sulfur cluster-dependent enzymes (complex I and II) involved in mitochondrial electron transport and cis-aconitase in the citric acid cycle. In addition, higher concentrations of NO,... 

The ribonucleotide reductases — a unique group of metalloenzymes essential for cell proliferation. M. Lammers and H. Follmann, Struct. Bonding (Berlin), 1983. 54, 27-91 (354). 

Thiobacillus ferrooxidans function. 6, 651 Rhus vernicifera stellacyanin structure, 6,651 Riboflavin 5 -phosphate zinc complexes, 5,958 Ribonucleotide reductases cobalt, 6,642 iron, 6,634... 

Labarre JF (1978) Conformational Analysis in Inorganic Chemistry Semi-Empirical Quantum Calculation vs. Experiment. 35 1-35 Lammers M, Follmann H (1983) The Ribonucleotide Reductases A Unique Group of Metalloenzymes Essential for Cell Proliferation. 54 27-91 Le Brun NE, Thomson AJ, Moore GR (1997) Metal Centres of Bacterioferritins of Non-Heam-Iron-Containing Cyrochromes 6557. 88 103-138... 

Sjbberg B-M (1997) Ribonucleotide Reductases - A Group of Enzymes with Different Metallosites and a Similar Reaction Mechanism. 88 139-174 Slebodnick C, Hamstra BJ, Pecoraro VL (1997) Modeling the Biological Chemistry of Vanadium Structural and Reactivity Studies Elucidating Biological Function. 89 51-108 Smit HHA, see Thiel RC (1993) 81 1-40... 

Sequence Comparison Between the N-Terminal Part of the Fepr Genes from Desulfovibrio desulfuricans (Dd) and Desulfovibrio vulgaris (Dv), Carbon Monoxide Dehydrogenase from Methanothrix soehngenii (Ms), Methanosarcina frisia Gdl (Mf), Clostridium thermoaceticum (Ct), Rhodospirillum rubrum (Rr), and Anaerobic Ribonucleotide Reductase from Escherichia coli (Ec) ... 

Rubrerythrin (Rr) was first isolated in 1988 from cellular extracts of D. vulgaris Hildenborough (38), and later also found in D. desulfuri-cans (39). Rr is constituted by two identical subunits of 22 kDa and it was shown that each monomer contains one Rd-like center, Fe(RS)4, and a diiron-oxo center similar to the ones found in methane monooxygenase (MMO) (40, 41) or ribonucleotide reductase (RNR-R2) (42). After aerobic purification, the UV-visible spectrum shows maxima at 492, 365, and 280 nm, and shoulders at 570 and 350 nm. This spectrum is similar to the ones observed for Rd proteins. From a simple subtraction of a typical Rd UV-vis spectrum (normalized to 492 nm) it is possible to show that the remainder of the spectrum (maxima at 365 nm and a shoulder at 460 nm) strongly resembles the spectrum of met-hemerythrin, another diiron-oxo containing protein. 

Adenosine deaminase deficiency is associated with an immunodeficiency disease in which both thymus-derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells) are sparse and dysfunctional. Purine nucleoside phosphorylase deficiency is associated with a severe deficiency of T cells but apparently normal B cell function. Immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby deplete cells of DNA precursors. 

Treatment via chelation has been observed for 2-acetylpyridine thiosemi-carbazone derivatives, which have been found to possess inhibitory activity for the RNA-polymerases of the influenza virus [133]. The iron(III) complexes were shown to be 3 to 6 times more active as inhibitors of partially purified ribonucleotide reductase (no added iron) compared to uncomplexed thiosemi-carbazone [128]. Raina and Srivastava [134] prepared and characterized low spin iron(III) complexes of 2-acetylpyridine thiosemicarbazone, [Fe(8-H)2A] (A = NO3, OH, Cl, N3, NCS or NO2), which were proposed as being seven-coordinate. However, all but the azide complex are 1 1 electrolytes in DMF and their solid ESR spectra are rhombic with the g-values being about 2.20,2.15 and 2.00. Of the six complexes, the azide ion seems to interact ihost strongly with the iron(III) center. 

The iron(III) complexes of 21 and 22 were shown to be 3 to 6 times more active as inhibitors of partially purified ribonucleotide reductase than un-complexed thiosemicarbazones [128]. The mechanism of antitumor action by these complexes still remains largely speculative, although some excellent preliminary studies have appeared. It has been postulated [148] that tridentate... 

Lammers, M., Follmann, H. The Ribonucleotide Reductases A Unique Group of Metalloenzymes Essential for Cell Proliferation. Vol. 54, pp. 27-91. 

The general influence of covalency can be qualitatively explained in a very basic MO scheme. For example, we may consider the p-oxo Fe(III) dimers that are encountered in inorganic complexes and nonheme iron proteins, such as ribonucleotide reductase. In spite of a half-filled crystal-field model), the ferric high-spin ions show quadrupole splittings as large as 2.45 mm s < 0, 5 = 0.53 mm s 4.2-77 K) [61, 62]. This is explained... 

Hydroxyurea is a ribonucleotide reductase inhibitor that prevents DNA synthesis and traditionally has been used in chemotherapy regimens. Studies in the 1990s also found that hydroxyurea increases HbF levels as well as increasing the number of HbF-containing reticulocytes and intracellular HbF. Other beneficial effects of hydroxyurea include antioxidant properties, reduction of neutrophils and monocytes, increased intracellular water content leading to increased red cell deformability, decreased red cell adhesion to endothelium, and increased levels of nitric oxide, which is a regulator involved in physiologic disturbances.22... 

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