Ribonucleotide reduction reductase

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... 

It was a surprise to discover that a mutant of E. coli lacking thioredoxin can still reduce ribonucleotides. In the mutant cells thioredoxin is replaced by glutaredoxin, whose active site disulfide linkage is reduced by glutathione rather than directly by NADPH. Oxidized glutathione is, in turn, reduced by NADPH and glutathione reductase. Thus, the end result is the same with respect to ribonucleotide reduction. 

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

Mao, S. S., Yu, G. X., Chalfoun, D., and Stubbe, J., 1992, Characterization of C439SR1, a mutant of Escherichia coli ribonucleotide diphosphate reductase evidence that C439 is a residue essential for nucleotide reduction and C439SR1 is a protein possessing novel thioredoxin-like activity, Biochemistry 31 9752n9759. 

The observation that these deoxyribonucleotide precursors exist in very low concentration in cells suggests that ribonucleotide reduction is a crucial and rate-controlling step in DNA synthesis and cell division. Indeed the level of reductase activity has been correlated with DNA replication (7), cell proliferation (2) and tumor growth rate (3, 4). 

The reduction of ribonucleotides to deoxyribonucleotides is linked to NADPH by thioredoxin and thioredoxin reductase. Thioredoxin is a small protein which is oxidized from a dithiol to a disulfide form during ribonucleotide reduction. The dithiol form of thioredoxin is regenerated by NADPH and a specific flavoprotein, thioredoxin reductase. The thioredoxin system consisting of thioredoxin and thioredoxin reductase was first identified as the reducing system from E. coli by Reichard and co-workers (35, 36) and both proteins have since been purified to homogeneity (37, 38). 

Fig. 10. Hydrogen donor systems for ribonucleotide reduction. Enzyme reactions are I thioredoxin reductase (EC 1.6.4.5) II ribonucleotide reductase (EC 1.17.4) III glutathione reductase (EC 1.6.4.2). GSH, GSSG reduced and oxidized glutathione NADPH, NADP reduced and oxidized nicotinamide adenine dinucleotide phosphate coenzymes. The hydrogen transfer chain is continued in Fig. II...

A huge amount of data, often still fragmentary, on allosteric regulation of all different types of ribonucleotide reductases awaits unambiguous analyses as the ones described. It is certainly not overstating to say that, next to the chemistry of ribonucleotide reduction, the enzymes interaction with nucleotides and the physiologic consequences is one of the most peculiar pieces of biochemistry. 

All eukaryotic ribonucleotide reductases assayed so far in vitro are inhibited by hydroxyurea, and a wide variety of eukaryotic cells and organisms is found susceptible to DNA synthesis inhibition by the drug . Nevertheless, a clearcut dependency like in E. coli cannot be stated as investigation of the eukaryotic enzymes is lagging and because hydroxyurea exerts manifold cellular effects, some without obvious relation to the inhibition of ribonucleotide reduction (cf. Table 9). 

Unspecific inhibition of ribonucleotide reduction is produced by compounds like pyridoxal phosphate, or the sulfonated anthraquinone-triazine dye, Cibacron blue. They interact, like in many enzymes, with nucleotide binding domains where pyridoxal phosphate becomes covalently linked to lysine, or in that the dye occupies the whole nucleotide fold. The latter interaction permits its use in affinity chromatography of ribonucleotide reductases Likewise, EDTA is not a specific, nor a potent inhibitor, it may, for example, act by complexation of the structure-stabilizing Mg " ions in native holoenzymes. However the iron-promoted radical regeneration process appears far more susceptible to interference from EDTA ... 

The initiation or arrest of enzyme synthesis responsible for the peak of ribonucleotide reductase is discussed below. Other factors which may modulate the activity in vivo or when measured in crude homogenates include allosteric control by the endogenous deoxyribonucleotides, the action of late S phase-specific inhibitors like the one found in Achlya, or the redox status of thioredoxin and glutaredoxin however the cell cycle dependence of these reactions is little known. It is therefore desirable to assay ribonucleotide reductase in preparations which have been subjected to at least one precipitation and dialysis step. While reliable cell cycle-dependent activity data are thus obtained, the absolute figures are frequently an order of magnitude too low to account for the cell s need of ribonucleotide reduction for DNA synthesis. This unsatisfactory condition is most likely a problem of quinary enzyme structure (see below) but is not felt to invalidate the accumulated evidence for tight correlation of ribonucleotide reduction and the cell cycle as a whole. 

Reichard for allosteric regulation of ribonucleotide reduction. One has to conclude that replitase-associated ribonucleotide reductase remains freely accessible for effectors from outside ... 

Reduction of ATP to the 2 -deoxy-compound has been carried out on a preparative scale using recombinant Lactobacillus leichmannii ribonucleotide triphosphate reductase. ... 

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