Enzyme ribonucleotide reductase

Theorists of the RNA World have debated whether the constituents of the cell arose in the sequence RNA-DNA-Protein or RNA-Protein-DNA. The universal use of ribonucleotide reductase enzymes provided an answer to this question. Can you see why ... 

Fig. 11. Hydrogen transfer from thioredoxin or glutaredoxin (R) to the ribonucleotide reductase enzyme protein ( ), radical intermediates (.Y), and nucleotides (partial structures, right)...

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.)...

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,... 

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... 

Non-heme Di-Iron Enzymes Methane Monooxygenase and Ribonucleotide Reductase... 

Metalloenzymes with non-heme di-iron centers in which the two irons are bridged by an oxide (or a hydroxide) and carboxylate ligands (glutamate or aspartate) constitute an important class of enzymes. Two of these enzymes, methane monooxygenase (MMO) and ribonucleotide reductase (RNR) have very similar di-iron active sites, located in the subunits MMOH and R2 respectively. Despite their structural similarity, these metal centers catalyze very different chemical reactions. We have studied the enzymatic mechanisms of these enzymes to understand what determines their catalytic activity [24, 25, 39-41]. 

A common way to benefit from the ability to combine different molecular orbital methods in ONIOM is to combine a DFT or ab-initio description of the reactive region with a semi-empirical treatment of the immediate protein environment, including up to 1000 atoms. Due to the requirement for reliable semi-empirical parameters, as discussed in Section 2.2.1, this approach has primarily been used for non-metal or Zn-enzymes. Examples include human stromelysin-1 [83], carboxypeptidase [84], ribonucleotide reductase (substrate reaction) [85], farnesyl transferase [86] and cytosine deaminase [87], Combining two ab-initio methods of different accuracy is not common in biocatalysis applications, and one example from is an ONIOM (MP2 HF) study of catechol O-methyltransferase [88],... 

Ribonucleotide reductase differs from the other 5 -deoxyadenosyl-cobalamin requiring enzymes in a number of respects. Hydrogen is transferred from coenzyme to the C2-position of the ribose moiety without inversion of configuration. Also since lipoic acid functions in hydrogen transfer, exchange with solvent protons takes place. Furthermore, exchange between free and bound 5 -deoxyadenosylcobalamin occurs rapidly during catalysis. Evidence for a Co(I)-corrin as an intermediate for this reduction is presented in our section on electron spin resonance. 

From these data it seems feasible that a Co(II)-species is generated during catalysis, and that homolysis of the Co—C-bond is a prerequisite for enzyme catalysis in ribonucleotide reductase. However, the kinetics of appearance of the Co(II)-signal indicates that the rate of formation of Co(II) is much slower than either the rate of ribonucleotide reduction... 

The application of ESR to the ribonucleotide reductase system indicates that the catalytic intermediate is a Co(I)-species. The appearance of Cob(Il)alamin is probably caused by partial oxidation of the Co(I)-species. In the enzyme bound Co(II)-species the benzimidazole ligand is coordinated, and the corrin ring is bound so tightly that the enzyme produces a unique highly resolved ESR spectrum. 

The application of magnetic resonance techniques to biological systems is a relatively new approach for the study of macromolecules. In this review we have presented the different approaches which have been made to study Bi2-enzymes. Clearly some progress has been made particularly from the application of ESR to a study of the enzymes ethanolamine ammonia-lyase and ribonucleotide reductase. Although 13C NMR is well in its developmental stages it is obvious that this technique will prove to be very useful for the examination of coenzyme-enzyme interactions. Studies of how corrinoids bind in enzymes and how sulfhydryl containing proteins are involved in enzyme catalysis comprise two major problems which must be overcome before realistic mechanisms can be presented for this group of enzymes. 

HU is an inhibitor of ribonucleotide reductase, a rate-limiting enzyme which catalyzes the conversion of ribonucleotides into deoxyribonucleotides. HU is thus a cytotoxic agent as it has the ability to inhibit DNA synthesis. Consequently, H U can affect only cells that are actively synthesizing DNA and, therefore, a drug of S-phase cell-cycle specific. Moreover, HU-mediated inhibition of ribonucleotide reductase is reversible, implying that the action of HU will exhibit a relatively straight forward concentration-time course dependence [2—4-]. 

Ribonucleotide reductase works on ribo-A, -U, -G, -C diphosphates to give the deoxynucleotide. The deoxyuridine, which is useless for RNA synthesis, is converted to deoxythymidine by the enzyme thymidylate synthase, which uses methylene tetrahydrofolate as a one-carbon donor. The odd thing here is that ribonucleotide reductase uses the UDP as a substrate to give the dUDP. This must then be hydrolyzed to the dUMP before thymidylate synthase will use it to make dTMP. Then the dTMP has to be kinased (phosphorylated) up to dTTP before DNA can be made. 

Domino reactions are not only useful for the construction of molecules, but also for their degradation. This concept is often encountered in nature. Thus, ribonucleotide reductases (RNRs) are enzymes that catalyze the formation of DNA monomers from ribonucleotides by radical mediated 2 -deoxygenation. This process has also been studied... 

Interest in this class of coordination compounds was sparked and fueled by the discovery that radical cofactors such as tyrosyl radicals play an important role in a rapidly growing number of metalloproteins. Thus, in 1972 Ehrenberg and Reichard (1) discovered that the R2 subunit of ribonucleotide reductase, a non-heme metal-loprotein, contains an uncoordinated, very stable tyrosyl radical in its active site. In contrast, Whittaker and Whittaker (2) showed that the active site of the copper containing enzyme galactose oxidase (GO) contains a radical cofactor where a Cu(II) ion is coordinated to a tyrosyl radical. 

The realization of the widespread occurrence of amino acid radicals in enzyme catalysis is recent and has been documented in several reviews (52-61). Among the catalytically essential redox-active amino acids glycyl [e.g., anaerobic class III ribonucleotide reductase (62) and pyruvate formate lyase (63-65)], tryptophanyl [e.g., cytochrome peroxidase (66-68)], cysteinyl [class I and II ribonucleotide reductase (60)], tyrosyl [e.g., class I ribonucleotide reductase (69-71), photosystem II (72, 73), prostaglandin H synthase (74-78)], and modified tyrosyl [e.g., cytochrome c oxidase (79, 80), galactose oxidase (81), glyoxal oxidase (82)] are the most prevalent. The redox potentials of these protein residues are well within the realm of those achievable by biological oxidants. These redox enzymes have emerged as a distinct class of proteins of considerable interest and research activity. 

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