Deoxyribonucleoside, synthesis, enzymes

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

DNA polymerase I is a nonessential enzyme, since viable E. coli mutants lack it (pol A). This conclusion is complicated, however, since the enzyme catalyzes three separate chemical reactions. It polymerizes deoxyribonucleoside triphosphates, and it has two exonucleolytic activities, a 3 to 5 activity and a 5 to 3 activity. The pol A - mutants lack only the polymerization activity. Other mutants lacking both the polymerase and the 5 to 3 exonuclease activity are lethal. Thus the exonuclease function is the more important one. This fits with the role of this enzyme in removing damaged DNA segments (DNA repair) and in removing covalently attached RNA from DNA chains. We will later see that small RNAs serve as primers of DNA synthesis. 

Biochemical Retrosynthesis of 2 -Deoxyribonucleosides from Glucose Acetaldehyde and a Nucleobase Three-Step Multi-Enzyme-Catalyzed Synthesis... 

Purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) catalyzes the reversible phosphorylysis of ribonucleosides and 2 -deoxyribonucleosides of guanine, hypoxanthine, and related nucleoside analogs [1]. It normally acts in the phosphorolytic direction in intact cells, although the isolated enzyme catalyzes the nucleoside synthesis under equilibrium conditions. Figure 1 shows the chemical reaction. 

Gemcitabine is phosphorylated initially by the enzyme deoxycytidine kinase and then by other nucleoside kinases to the di- and triphosphate nucleotide forms, which then inhibit DNA synthesis. Inhibition is considered to result from two actions inhibition of ribonucleotide reductase by gemcitabine diphosphate, which reduces the level of deoxyribonucleoside triphosphates required for the synthesis of DNA and incorporation of gemcitabine triphosphate into DNA. Following incorporation of gemcitabine nucleotide, only one additional nucleotide can be added to the growing DNA strand, resulting in chain termination. 

For DNA synthesis, all four deoxyribonucleoside 5 -triphos-phates (dATP, dGTP, dCTP and dTTP) must be present, as well as Mg2+, a primer molecule ((deoxynucleotide)n shown in Equation 1) and a DNA template. A detailed mechanism of the reaction must consider that the enzyme binds at least four compounds simultaneously, these being a deoxynucleoside 5 -triphosphate, Mg2+, a deoxynucleotide primer molecule and a DNA template. The enzyme is thus able to convert an impossible five component reactant system into a unimolecular reaction, contributing substantially to the efficiency of the catalyzed reaction. 

Regulation of ribonucleotide reductase is complex. The binding of dATP (deoxyadenosine triphosphate) to a regulatory site on the enzyme decreases catalytic activity. The binding of deoxyribonucleoside triphosphates to several other enzyme sites alters substrate specificity so that there are differential increases in the concentrations of each of the deoxyribonucleotides. This latter process balances the production of the 2 -deoxyribonucleotides required for cellular processes, especially that of DNA synthesis. 

These enzymic reactions are essential to all living cells in that they provide the monomeric precursors for the de novo synthesis of DNA. The production of the 2 -deoxyribonucleoside phosphates required for DNA synthesis is carefully regulated through allosteric control of the enzyme by 2 -deoxynucleoside triphosphates and ATP, which regulate both overall activity and substrate specificity. 

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. 

Hydroxyurea inhibits ribonucleotide reductase. By sequestering ferric ions, hydroxyurea destabilizes the organic bee radical in the R2 subunit of the enzyme. The inhibition of enzyme activity leads to a depletion of deoxyribonucleoside diphosphates, which are normally converted to deoxyribonucleoside triphosphates, the substrates for DNA synthesis. 

The known in-vitro properties of DNA polymerizing enzymes do not provide an explanation of the in-vivo initiation of DNA synthesis (Kornberg, 1969 DeLucia and Cairns, 1969). Consider in-vitro polymerization of deoxyribonucleoside triphosphates by E, coli DNA polymerase. This enzyme catalyzes the addition of deoxyribonucleoside triphosphates to the 3 -hydroxyl terminus of a primer DNA. Such synthesis occurs only in the direction of 5 to 3 , and in all cases studied there is an absolute requirement for DNA template (Kornberg, 1969). The action of E, coli polymerase is illustrated in Figure 4. A DNA template must have an available 3 -hydroxyl terminated strand which can serve as a primer for the initiation of synthesis, and it is assumed that polymerase is bound to an area of the template strand near the 3 -end of the primer. The deoxynucleoside triphosphate is bound adjacent to the 3 -hydroxyl group of the terminal nucleotide to form a base pair with the template. When the correct base pair is formed the polymerase catalyzes a nucleophilic attack by the 3 -hydroxyl group of the primer on the a-phosphorus of the triphosphate. A phosphodiester bond is formed with the subsequent release of pyrophosphate (Fig. 4). 

PSF specifically stimulated the primase activity and, thus, the DNA replicase activity of purified DNA polymerase a-primase. As shown in Fig. 1, the primer synthesis by this enzyme was completely dependent on template, required appropriate concentration of deoxyribonucleoside triphosphate and was markedly stimulated by a very low concentration of PSF (more than 20-fold stimulation was observed at 10 ng/50 pi of PSF). The factor was also effective in stimulating the replicase activity supported by single stranded calf thymus or 0X 174 DNA as template, indicating that primer synthesis in these reactions was also stimulated. However, PSF is not primase itself since the factor neither shows any primer synthesis by itself nor does it stimulate the activity of E. coli DNA polymerase I with poly (dT) as template. A factor with properties similar to PSF has been found in mouse tissue by Yagura, Kozu, and Seno (8) but was not detected in other vertebrates tested by them (9). However, the molecular component of their factor was significantly smaller (63 kDa) than the one described here (8). 

These enzymes are used for both the utilization and synthesis of deoxyribonucleosides. In the catabolic utilization of the nucleosides as carbon sources, they function in concert with two other enzymes, deoxyribose-1-phosphate mutase drm) and deox3rribose-5-phosphate aldolase drd). The enzymes under consideration and their reactions are summarized as follows. 

Enzymic methods of nucleoside i thesis continue to attract attention, and the sjmthesis of radiolabelled ribo- and deoxyribonucleosides by transglycosylation catalysed by crude E.coU homogenates has been reviewed.27 A promising method for the enzymic synthesis of nucleosides is illustrated by the example of virazole synthesis in Scheme 1 the use of A/7-methyl guanosine... 

The 2-deoxy-D-ribose 5-phosphate aldolase (RibA or DERA EC 4.1.2.4) is a class I enzyme that, in vivo, catalyzes the reversible addition of acetaldehyde to D-glyceraldehyde 3-phosphate (34 Figure 5.57) in the metabolic degradation of 127 from deoxyribonucleosides [269], ivith an equilibrium constant for synthesis of 2 x lO m [56]. It is, therefore, unique among the aldolases in that it uses an aldehyde rather than a ketone as the aldol donor. RibA has been isolated from eukaryotic and prokaryotic sources [270, 271],... 

Enzymes have been isolated from the most diverse sources that can support DNA synthesis in a cell-free system. They are called DNA polymerases or DNA replicases. They need, in addition to other cofactors, DNA as primer and deoxyribonucleoside-5 -triphosphates as DNA building blocks. The triphosphates align themselves on the DNA single strand introduced as primer according to the rules of base pairing an... 

The enzymic propanoylation of 2-deoxy-D-ribose at 0-5 is the first step in a new one-pot chemicoenzymatic synthesis of 2 -deoxyribonucleosides. Alginate gel-entrapped cells of an auxotrophic thymine-dependent strain of E. coli have been used to catalyse the transfer of the 2-deoxy-D-ribofuranosyl unit from T-deoxyuridine to purine and pyrimidine bases, as well as to their aza- and deaza-analogues. Enzymic transglycosylation was also used as a stereoselective alternative to chemical synthesis in the preparation of the imidazole deoxynucleo-side 22.36... 

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