Nucleoside diphosphate reductase

DNA and RNA synthesis De novo formation of purine and pyrimidine nucleotide Nucleoside diphosphate reductase Thymidylate synthase Polymerase reactions Chapter 20... 

The synthesis of purine nucleotides (1) starts from IMP. The base it contains, hypoxanthine, is converted in two steps each into adenine or guanine. The nucleoside monophosphates AMP and CMP that are formed are then phos-phorylated by nucleoside phosphate kinases to yield the diphosphates ADP and GDP, and these are finally phosphorylated into the triphosphates ATP and CTP. The nucleoside triphosphates serve as components for RNA, or function as coenzymes (see p. 106). Conversion of the ribonucleotides into deoxyribo-nucleotides occurs at the level of the diphosphates and is catalyzed by nucleoside diphosphate reductase (B). 

In the first step, thioredoxin reductase reduces a small redox protein, thioredoxin, via enzyme-bound FAD. This involves cleavage of a disulfide bond in thioredoxin. The resulting SH groups in turn reduce a catalytically active disulfide bond in nucleoside diphosphate reductase ( ribonucleotide reductase ). The free SH groups formed in this way are the actual electron donors for the reduction of ribonucleotide diphosphates. 

Reducing equivalents from a ven thioredoxin can be donated to a variety of reductase enzymes. They are not specific for the nucleotide reductase or for enzymes from the same organism. Reduced yeast thioredoxin will serve as reductant for methionine sulphoxide reductase, sulphate reductase and the E. coli nucleoside diphosphate reductase. Heat-stable protein cofactors are known to be involved in each of these systems. 

Deoxynucleotides for DNA synthesis are made at the nucleoside diphosphate level and then have to be phosphorylated up to the triphosphate using a kinase and ATP. The reducing equivalents for the reaction come from a small protein, thioredoxin, that contains an active site with two cysteine residues. Upon reduction of the ribose to the 2 -deoxyri-bose, the thioredoxin is oxidized to the disulfide. The thioredoxin(SS) made during the reaction is recycled by reduction with NADPH by the enzyme thioredoxin reductase. 

ADP as a substrate in enzyme reactions, ADENYLATE KINASE (or MYOKINASE) ATP SYNTHASE CREATINE KINASE NUCLEOSIDE DIPHOSPHATE KINASE PHOSPHOGLYCERATE KINASE PYRUVATE KINASE RIBONUCLEOTIDE REDUCTASE SULFATE ADENYLYLTRANSFERASE (ADP) [ADP]/[ATP] ratio,... 

Fluoro and 3, 3-difluoromethylene nucleosides have been prepared as inhibitors of ribonucleotide diphosphate reductase (cf. Chapter 7). ... 

Ribonucleotide reductase ribonucleoside diphosphate reductase) is a multisubunit enzyme (two identical B1 subunits and two identical B2 subunits) that is specific for the reduction of nucleoside diphosphates (ADP, GDP, CDP, and UDP) to their deoxy-forms (dADP, dGDP, dCDP, and dUDP). The immediate donors of the hydrogen atoms needed for the reduction of the 2-hydroxyl group are two sulfhydryl groups on the enzyme itself, which, during the reaction, form a disulfide bond (Figure 22.12). 

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. 

The enzyme contains two catalytic sites, two regulatory sites and two specificity sites. The catalytic site binds the substrates, thioredoxin (reduced by NADPH + H+) and the nucleoside diphosphates. The allosteric regulatory site binds ATP as an activator in competition with dATP as an inhibitor. The specificity site binds dGTP, dTTP and dATP but not dCTP and modulates ribonucleotide reductase activity selectively for the four NDP substrates to balance the four dNTP pools. 

B) The patterns of regulation with regard to different nucleoside diphosphates demonstrated by ribonucleotide reductase. 

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 reductase detected in crude extracts of this fungus showed barely detectable activity with purine nucleoside diphosphates and was most active with ATP and GTP (77). 

These compounds cause 50% inhibition of a partially purified reductase from Novikoff rat tumor at 10 8 to 10-7 M (150,151). Approximately the same degree of inhibition was observed with other mammalian reductases (152, 153), but the non-heme iron containing reductase from E. coli was not affected. The inhibition of the mammalian reductases is only partially reversible (154). Since these compounds are strong metal chelators complexation of iron is probably involved in the mechanism of inhibition however excess Fe2+ does not reverse the inhibition, and other evidence indicates that these compounds do not act solely by chelating free iron from solution thus depriving the enzyme of a cofactor (150, 151). Kinetic studies indicate no competition with respect to nucleoside diphosphate substrate, nucleotide effector, or magnesium ions, but partial competition for the dithiol substrate was observed. 

Deoxy-2 -fluoromethylene nucleosides are potential inhibitors of ribonucleoside diphosphate reductase (RDPR), which catalyzes the essential biosynthesis path of deoxyri-bonucleoside in the DNA synthesis system. Initial hydrogen abstraction at the C-3 position by tyrosyl radical has been proposed for the RDPR catalysis system (see from 71 to 73 in... 

Class I E. coli ribonucleotide reductase (RNR) exploits all the PCET variances of Fig. 17.3 in order to catalyze the reduction of nucleoside diphosphates to deoxynu-cleoside diphosphates. This reaction demands radical transport across two subunits and over a remarkable 35 A distance [187,188, 219]. The crystal structures of both R1 and R2 subunits have been solved independently [220-222] and a docking model has been proposed [220]. R2 harbors the diferric tyrosyl radical ( Y122) cofactor that initiates nucleotide reduction by generating a transient thiyl radical ( C439) in the enzyme active site located >35 A away in R1 [223]. Substrate conversion is initiated by a hydrogen atom abstraction (Type A PCET) at the 3 position of the substrate by C439 [192]. 

Harwood, H.J. Brandt, K.G. Rodwell, V.W. Allosteric activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by nucleoside diphosphates. J. Biol. Chem., 259, 2810-2815 (1984)... 

In a study related to the mechanism of inactivation of ribonucleotide diphosphate reductase (RDPR) by 2 -deoxy-2 -substituted nucleotide analogues, it was found that treatment of nucleosides of type 57 (B=Ura, Ade, X=I, Br, Cl, SMe, N3) with tributylstannane and AIBN gave the d4 products 58, whilst with X=F, OMs or OTs, the 3 -deoxysystem 59 was the product. The results pointed to the loss of radicals rather than anions from C-2 during mechanism-based inactivation of RDPR, and the authors suggest some modifications to Stubbe s mechanistic proposals. ... 

Acyl-CoA synthetase Cytochrome b-5 Cytochrome c reductase Kynurenine hydroxylase Monoamine oxidase Nucleoside diphosphate kinase Intermembrane space Adenylate kinase Inner boundary membrane... 

The kinetic mechanism of inhibition of ribonucleoside diphosphate reductase by a-(AO-heterocyclic carboxaldehyde thiosemicarbazones is not clear. The concentrations of the nucleoside diphosphate substrate, the allosteric activator ATP, or magnesium ion do not influence the inhibition of the enzyme produced by all of the thiosemicarbazones tested to date. Interesting differences exist, however, between the ring hydroxylated and nonhydroxylated a-(AO-heterocyclic carbox-... 

The ability of nucleoside triphosphates, singly, or in combination, to effect changes in the catalytic properties of the reductase have been explored systematically by Reichard and his co-workers the complicated pattern of results is summarized in Table 16-11. These findings have been interpreted in terms of four states of activity for the reductase. In the presence of ATP, the reductase prefers as substrates pyrimidine nucleoside diphosphates. In the presence of dGTP, a purine-specific state is assumed and dTTP causes the enzyme to be in a condition in which both purine and pyrimidine ribonucleoside diphosphates are reduced. 

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