Mechanism of ribonucleotide reductases

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. 

Kolberg, M., Strand, K.R., Graff, P. and Andersson, K.K. (2004) Structure, function and mechanism of ribonucleotide reductases, Biochim. Biophys. Acta, 1699, 1-34. 

Siegbahn, P. E. M., 1998, Theoretical study of die substote mechanism of ribonucleotide reductase J. Amer. Chem. Soc. 120 8417n8439. 

Figure 5 Proposed catal)dic mechanism of ribonucleotide reductase. (Reprinted with permission from Ref. 34. Chemical Society)...

The complex structure and molecular mechanism of ribonucleotide reductases make them vulnerable to many kinds of distortion. Not only has it been an. . experience that T 4 ribonucleotide reductase cannot stand a 0.5 M concentration of anything except water but enzymes from all sources are easily inhibited by one or several of the following interferences ... 

Sjoberg B-M, Sahlin M. 2002. Thiols in redox mechanism of ribonucleotide reductase. In Protein sensors and reactive oxygen species, Pt B, thiol enzymes and proteins, pp. 1-21. Heidelberg Elsevier Science. 

Figure 13.4 A proposed mechanism for all three classes of ribonucleotide reductases. Classes I and II RNRs require an active site Glu residue and a pair of redox-active Cys. Class HI RNRs lack the Glu and one of the Cys, and use formate as the reductant. (From Stubbe etal., 2001. Copyright 2001, with permission from Elsevier.)...

MECHANISM FIGURE 22-41 Proposed mechanism for ribonucleotide reductase. In the enzyme of . coli and most eukaryotes, the active thiol groups are on the R1 subunit the active-site radical (—X ) is on the R2 subunit and in . coli is probably a thiyl radical of Cys439 (see Fig. 22-40). Steps (T) through are described in the text. 

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. 

Hydroxyurea also inhibits DNA synthesis by its action on the M2 subunit of ribonucleotide reductase, but in this case it is the reduction of the purine nucleoside diphosphates which is inhibited and the pool of dTTP rises slightly (Turner et al., 1966 Adams and Lindsay, 1967 Krakoff et al., 1968 Adams et al., 1971 Skoog and Bjursell, 1974 Thelander et al., 1984). What prevents the pool rising dramatically is not clear, but some mechanism comes into play to reduce turnover of the dTTP pool (Nicander and Reichard, 1985). Its action is most satisfactorily reversed by changing the medium for drug free medium. 

Fig. 5. Mechanism of action of dinuclear non-haem iron enzymes utilising ferryl intermediates. Mechanisms for ribonucleotide reductase and methane mono-oxygenase adapted from that of Que [72]. Compound I and compound II define intermediates at the same oxidation state as the equivalent peroxidase intermediate (see Fig. 2). X is an unknown group suggested to bridge between the two iron atoms and form a cation radical. The nature of the electron required for the reduction of ribonucleotide reductase compound II is not clear - it is possible that this intermediate can also oxidise tyrosine [72].

Bollinger, J. M. J., Edmondson, D. E., Huynh, B. H., Eilley, J., Norton, J. R., and Stubbe, J., 1991, Mechanism of assembly of the tyrosyl radical-dinuclear iron cluster cofactor of ribonucleotide reductase. Science 253 292n298. 

An unprecedented example of the application of an organic azide as an enzyme inhibitor derives from the elegant studies of Stubbe and coworkers at MIT, who have investigated the mechanism of action of ribonucleotide reductase (RNR) using several mechanism-based inhibitors including 2 -azido-2 -deoxyuridine-5 -diphosphate (NjUDP) (71) [82]. RNR plays a... 

Mechanism A Tyrosyl Radical Is Critical to the Action of Ribonucleotide Reductase... 

T wo main classes of adenosylcobalamin-activated enzymes function by facilitating the homolytic scission of the Co-C5 to cob(II)alamin and the 5 -deoxyadenosyl radical. The resultant 5 -deoxyadenosyl radical initiates catalysis by abstraction of a hydrogen atom, either from a substrate in the case of the class of enzymes that catalyze radical isomerizations, or by abstraction of a hydrogen atom from Cys408-/3-SH in the active site of ribonucleotide reductase II. The resultant enzymatic thiyl radical initiates the reduction mechanism by abstraction of a hydrogen atom from the ribonucleotide substrate. We shall begin with the isomerization/elimination reactions of adenosylcobalamin. 

Ribonucleotide reductase is the enzyme that catalyzes synthesis of deoxyribonucleoside diphophosphates (dNDPs) from ribonucleoside diphosphates (rNDPs). Ribonucleotide reductase reduces the hydroxyl at carbon 2 of the ribose sugar in the rNDP to a hydrogen, forming a deoxyribose sugar and a corresponding dNDP. A free-radical mechanism is involved in the reaction. Three classes of ribonucleotide reductases are known. 

R1. It contains the active site. The two subunits make up the small subunit of the protein called R2, which contains the free radical. A clue to the mechanism of action of the enzyme (tyrosine free radical) is shown in Figure 22.14. Hydroxyurea, an inhibitor of ribonucleotide reductase, destroys the free radical. 

A proposed mechanism of action of ribonucleotide reductase is shown in Figure 22.15. Reduction of the ribonucleotides requires electrons. These ultimately come from NADPH and are delivered to ribonucleotide reductase by either thioredoxin or glutaredoxin, as shown in Figure 22.16. Evidence exists for a possible third electron carrier in E. coli. Some of the interesting biological activities of thioredoxin are listed in Table 22.1. 

Ribonucleotide reductase, the enzyme catalyzing the synthesis of dNDPs from rNDPs, reduces the hydroxyl at carbon 2 to a hydrogen via a free radical mechanism. The following three classes of ribonucleotide reductases are known ... 

Thioredoxin is also a potential carrier of electrons to ribonucleotide reductase for reduction of ribose to deoxyribose. A proposed mechanism of action of ribonucleotide reductase is shown in Figure 22.15. 

Purine deoxyribonucleotides are derived primarily from the respective ribonucleotide (Fig. 6.2). Intracellular concentrations of deoxyribonucleotides are very low compared to ribonucleotides usually about 1% that of ribonucleotides. Synthesis of deoxyribonucleotides is by enzymatic reduction of ribonucleotide-diphosphates by ribonucleotide reductase. One enzyme catalyzes the conversion of both purine and pyrimidine ribonucleotides and is subject to a complex control mechanism in which an excess of one deoxyribonucleotide compound inhibits the reduction of other ribonucleotides. Whereas the levels of the other enzymes involved with purine and pyrimidine metabolism remain relatively constant through the cell cycle, ribonucleotide reductase level changes with the cell cycle. The concentration of ribonucleotide reductase is very low in the cell except during S-phase when DNA is synthesized. While enzymatic pathways, such as kinases, exist for the salvage of pre-existing deoxyribosyl compounds, nearly all cells depend on the reduction of ribonucleotides for their deoxyribonucleotide... 

DNA synthesis depends on a balanced supply of the four deoxyribonucleotides [1]. In all living organisms, with no exception so far, this is achieved by reduction of the corresponding ribonucleotides (substrates can be either ribonucleoside diphosphates NDP or ribonucleoside triphosphates NTP) by NADPH (Scheme 10-1), through a complex free radical chemistry. The substrate specificity is modulated by a sophisticated allosteric mechanism which makes it possible for a single protein to regulate the reduction of all four conunon ribonucleotides. This aspect will not be discussed here. Three well-characterized classes of ribonucleotide reductases (RNRs) have been described so far, which all are radical metalloenzymes [2-5]. 

Describe the subunit structure of ribonucleotide reductase. Outline its reaction, mechanism, and regulation. Explain the roles of NADH, thioredoxin, and thioredoxin reductase in the reaction. 

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