Ribonucleotide reductase catalyzed reactions

The reactions catalyzed by B12 may be grouped into two classes those catalyzed by methylcobalamin and those catalyzed by cofactor B,2. The former reactions include formation of methionine from homocysteine, methanogenesis (formation of methylmercury is an important side reaction), and synthesis of acetate from carbon dioxide (82). The latter reactions include the ribonucleotide reductase reaction and a variety of isomerization reactions (82). Since dehydration and deamination have been studied quite extensively and very possibly proceed via [Pg.257]

One example of an enzyme-catalyzed reaction involving a radical intermediate is the enzyme ribonucleotide reductase, which catalyzes the conversion of ribonucleotides (used for RNA biosynthesis) to 2 -deoxyribonucleotides (used for DNA biosynthesis), as illustrated in Fig. 16. Spectroscopic studies of the R2 subunit of Escherichia coli ribonucleotide reductase have shown that it can form a stable, long-lived, tyrosyl radical species—the first protein radical to be discovered (13). 

The use of a mixed-valent, dinuclear iron site, similar to those in hemerythrin and ribonucleotide reductase,to catalyze a nonredox reaction such as phosphate ester hydrolysis is novel and unexpected for a variant of the familiar oxo(hydroxo)-bridged diiron center. In contrast to the general agreement that exists regarding the spectroscopic and physical properties of the PAPs, their kinetics properties and especially their mechanism of action remain controversial. Much of the disagreement stems from the different pH dependences of the catalytic activity of BSPAP and Uf, which is due to the fact that the former is isolated in a proteolytically activated form while the latter is not. Proteolysis results in a substantial increase in optimal pH in addition to an increase in catalytic activity at the optimal pH. "" Current data suggest that many of the spectroscopic studies described in the literature were performed on a catalytically inactive form of the enzyme. As a result, the roles of the trivalent and divalent metal ions in catalysis and in particular the identity of the nucleophilic hydroxide that directly attacks the phosphate ester remain unresolved. 

The most conspicuous use of iron in biological systems is in our blood, where the erythrocytes are filled with the oxygen-binding protein hemoglobin. The red color of blood is due to the iron atom bound to the heme group in hemoglobin. Similar heme-bound iron atoms are present in a number of proteins involved in electron-transfer reactions, notably cytochromes. A chemically more sophisticated use of iron is found in an enzyme, ribo nucleotide reductase, that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an important step in the synthesis of the building blocks of DNA. 

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]. 

Ribonucleotide reductase is responsible for the conversion of the four biological ribonucleotides (RNA) into their corresponding deoxy forms (DNA). Although RNR is not an oxygenase during its primary catalyzed reaction (the conversion of ribonucleotides), it activates oxygen to generate a stable tyrosyl radical that is essential to the overall mechanism [46 49]. The common link between the chemistry of MMO and RNR is the activation of O2 by the di-iron active site. 

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... 

Ribonucleotide reductase (diphosphate) [EC 1.17.4.1], also known as ribonucleoside-diphosphate reductase, catalyzes the reaction of a 2 -deoxyribonucleoside diphosphate with oxidized thioredoxin and water to produce a ribonucleoside diphosphate and reduced thioredoxin. This system requires the presence of iron ions and ATP. Ribonucleotide reductase (triphosphate) [EC... 

FIGURE 22-43 Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase. Figure 22-44 gives details of the thymidylate synthase reaction. 

An unusual feature of ribonucleotide reductase is that the reaction it catalyzes involves a radical mechanism. The mammalian type of reductase initiates this reaction by the tyrosyl radical-nonheme iron. Hydroxyurea and related inhibit the majTrrrraiVarr retftrcCase 6y abolishing the radical state of the tyrosine residue. Inhibition of DNA synthesis by such compounds is secondary to this effect. 

Scheme 2.6 Examples of reactions catalyzed byenzymesthat carry a dinudear iron active site (a) hydroxylation of methane by soluble methane monooxygenase (sMMO) [7] (b) reduction of ribonucleotides by class I ribonucleotide reductase (RNR)...

Figure 6 Reactions catalyzed by ribonucleotide reductase (RNR). HS-E-SH is the site of the enzyme responsible for the reduction step SH is a cysteine thiol group.

Glutaredoxin is another small ubiquitous protein with a different dithiol-active center which catalyzes GSH-disulfide transhydrogenase reactions. It is GSH-specific and cannot be reduced by thioredoxin reductase. It uses GSH and an NADPH-coupled glutaredoxin reductase to catalyze the reduction of a variety of disulfide substrates, including 2-hydroxyethyl-disulfide and ribonucleotide reductase [281]. Since GSSG inhibits the latter reaction, a high ratio of GSH to GSSG will promote the synthesis of deoxyribonucleotides, which is a likely control mechanism of DNA synthesis. 

The most comprehensive set of kinetic studies on AdoCbl homolysis have been performed with AdoCbl-dependent ribonucleotide reductase by Stubbe and coworkers (Licht et al., 1999a Licht et al., 1999b). In this enzyme, AdoCbl is used to generate a thiyl radical on a cysteine residue it is this thiyl radical that abstracts hydrogen from the 3-position of the ribonucleotide to faeilitate reduction at C-2. In the presence of dGTP, an allosteric activator of the enzyme, the enzyme catalyzes the reversible cleavage of AdoCbl and formation of thiyl radical in the absence of substrate. This partial reaction proceeds rapidly enough for it to be mechanistically relevant and provides a system to study AdoCbl homolysis which is both simple and amenable to detailed kinetic analysis. 

Logan, D. T, deMarE, F., Persson, B. O., Slaby, A., Sj berg, B.-M., and Nordlund, P., 1998, Crystal structures of two self-hydroxylating ribonucleotide reductase protein R2 mutants Structural basis for the oxygen-insertion step of hydroxylation reactions catalyzed by diiron proteins. Biochemistry 37 10798nl0807. 

The participation of a cysteine thiol in the abstraction of hydrogen from ribose C-3 coenzyme B12, which is not a feature of any other of the reactions of Table 1, leads to exchange of H between this thiol group, the 5 -methylene group of coenzyme Bi2 and water (22). It was also shown that ribonucleotide reductase catalyzes the conversion of adenosylcobalamin labeled with one deuterium atom at C-5 (initial R/S ratio = 3 1) to monodeuterated coenzyme with R/S ratio = 1. This result shows that the cobalt-carbon 0-bond is reversibly cleaved to a 5 -deoxyadenosyl radical, which permits rotation about the C-47C-5 0-bond. 

In chloroplasts, oxidized thioredoxin is reduced by ferredoxin in a reaction catalyzed by ferredoxin-thioredoxin reductase. This enzyme contains a 4Fe-4S cluster that couples two one-electron oxidations of reduced ferredoxin to the two-electron reduction of thioredoxin. Thus, the activities of the light and dark reactions ofphotosynthesis are coordinated through electron transfer from reduced ferredoxin to thioredoxin and then to component enzymes containing regulatory disulfide bonds (Figure 20.16). We shall return to thioredoxin when we consider the reduction of ribonucleotides (Section 25.3). 

Conversion of ribonucleotides to the deoxy forms occurs exclusively at the diphosphate level. Ribonucleo-side diphosphate reductase (ribonucleotide reductase) catalyzes the reaction. This enzyme is found in all species and tissues. The immediate source of reducing equivalents is the enzyme (E) itself in which two sulfhydryl groups are oxidized to a disulfide. The general reaction is... 

Ribonucleotide reductase activity was assayed based on CDP reduction, using a modified method of Jong et al. (1998), with the [ CICDP reduction product determined as radioactivity incorporated into DNA in a series of two coupled reactions, catalyzed by nucleoside diphosphate kinase and DNA polymerase (Klenow fragment). A 40 pi reaction mixture contained 50 mM Hepes pH 7.2, 10 mM dithiothreitol. 

The ribonucleotide reductase system, consisting of ribonucleotide reductase, thioredoxin and thioredoxin reductase, catalyzes the irreversible reduction of the four common ribonucleoside-5 -phosphates to the corresponding 2 -deoxyribonucleoside-5 -phosphates. This reaction provides the deoxy ribonucleotide precursors necessary for DNA synthesis. 

Ribonucleotide reductase 19,37-41) catalyzes a highly regulated essential reaction for all living cells, the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides. The iron-containing class I RNR is found in some bacteria and eukaryotic cells, and it is... 

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