Ribonucleotide reductase reaction mechanisms

A likely mechanism for the ribonucleotide reductase reaction is illustrated in Figure 22-41. The 3 -ribonu-cleotide radical formed in step (T) helps stabilize the cation formed at the 2 carbon after the loss of H20 (steps and (3)). Two one-electron transfers accompanied by oxidation of the dithiol reduce the radical cation (step ). Step (5) is the reverse of step ( ) regenerating the active site radical (ultimately, the tyrosyl radical) and forming the deoxy product. The oxidized dithiol is reduced to complete the cycle (step ). Ini , coli, likely sources of the required reducing equivalents for this reaction are thioredoxin and glutaredoxin, as noted above. 

Abeles, R. H., and Beck, W. S., 1967, The mechanism of action of cobamide coenz>me in the ribonucleotide reductase reaction. J. Biol. Chem. 242 3589fi3593. 

The mechanism proposed in Scheme 5 makes experimentally verifiable predictions concerning the mechanism of the ribonucleotide reductase reaction with normal substrates and substrate analogs. Two crucial predictions are the cleavage of the 3 -carbon-hydrogen bond and the return of (possibly the same) hydrogen to the 3 -position on the same face from which the original atom was abstracted. 

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... 

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. 

Ribonucleotide reductase is notable in that its reaction mechanism provides the best-characterized example of the involvement of free radicals in biochemical transformations, once thought to be rare in biological systems. The enzyme in E. coli and most eukaryotes is a dimer, with subunits designated R1 and R2 (Fig. 22-40). The R1 subunit contains two lands of regulatory sites, as described below. The two active sites of the enzyme are formed at the interface between the R1 and R2 subunits. At each active site, R1 contributes two sulfhydryl groups required for activity and R2 contributes a stable tyrosyl radical. The R2 subunit also has a binuclear iron (Fe3+) cofactor that helps generate and stabilize the tyrosyl radicals (Fig. 22-40). The tyrosyl radical is too far from the active site to interact directly with the site, but it generates another radical at the active site that functions in catalysis. 

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. 

Cho KB, V Pelmenschikov, A Graslund, PEM Siegbahn (2004) Density functional calculations on class III ribonucleotide reductase Substrate reaction mechanism with two formates. J. Phys. Chem. B 108 (6) 2056-2065... 

Torrent, M., Musaev, D. G., Basch, H. and Morokuma, K. (2002) Computational studies of reaction mechanisms of methane monooxygenase and ribonucleotide reductase, J. Comput. Chem. 23, 59-76. 

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. 

Bollinger, J. M., Tong, W. H., Ravi, N., Huynh, B. H., Edmondson, D. E., and Stuhhe, J., 1994h, Mechanism of assembly of the tyrosyl radical-diiron(III) cofactor of E. coli ribonucleotide reductase III. Kinetics of the limiting Fe reaction by optical, EPR, and M ssbauer spectroscopies. /. Am. Chem. Soc. 116 8024n8032. 

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. 

We turn now to the synthesis of deoxyribonucleotides. These precursors of DNA arc formed by the reduction ot ribonucleotides specifically the 2 -hydroxyl group on the ribose moiety is replaced by a hydrogen atom. The substrates are ribonucleoside diphosphates, and the ultimate reduclant is NADPH. The enzyme ribonucleotide reductase is responsible for the reduction reaction for all four ribonucleotides. The ribonucleotide reductases of different organisms are a remarkably diverse set of enzymes. Yet detailed studies have revealed that they have a common reaction mechanism, and their three-dimensional structural features indicate that these enzymes are homologous. We will focus on the best understood of these enzymes, that of E. coli living aerobically. 

Since 1976 a number of 2 -substituted 2 -deoxynucleoside 5 -diphosphates have been shown to be mechanism-based inactivators of the ribonucleotide reductases from a variety of sources (Table I). This work was extended by Stubbe and Kozarich 50, 51), who studied the reaction of several 2 -halo-substituted 2 -deoxynucleoside 5 -diphosphates with RDPR. Incubation of RDPR with CIUDP, 2 -deoxy-2 -fluoroadenosine 5 -diphosphate (FADP), or 2 -deoxy-2 -fluorocytidine 5 -diphosphate (FCDP) resulted in time-dependent enzyme inactivation, concomitant and stoichiometric loss of all substituents from the ribose moiety, increase in the UV-Vis absorbance of the protein near 320 nm, and... 

The proposed mechanisms for the utilization of substrates and substrate analogs by the ribonucleotide reductases. Schemes 5 and 6, predict the formation of various radical intermediates. To search for these intermediates and their direct participation in these reactions, EPR spectroscopy has been employed (60). However, these attempts have not revealed the generation of new radical signals. 

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