Deoxyribonucleoside phosphorylases

Enzymes cataboiising deoxyribonucieosides Deoxyribomutase Deoxyriboaldolase Purine deoxyribonucleoside phosphorylase... 

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. 

The reaction catalyzed by polynucleotide phosphorylase differs fundamentally from the polymerase activities discussed so far in that it is not template-dependent. The enzyme uses the 5 -diphosphates of ribonucleosides as substrates and cannot act on the homologous 5 -triphos-phates or on deoxyribonucleoside 5 -diphosphates. The RNA polymer formed by polynucleotide phosphorylase contains the usual 3, 5 -phosphodiester linkages, which can be hydrolyzed by ribonuclease. The reaction is readily reversible and can be pushed in the direction of breakdown of the polyribonucleotide by increasing the phosphate concentration. The probable function of this enzyme in the cell is the degradation of mRNAs to nucleoside diphosphates. 

Such a catabolic reaction is indeed excluded in 6 alkyl purine derivatives. The parent compound of this group, 6-methylpurine, is known for its cytotoxicity its libera tion from the 2 -deoxyribonucleoside by purine nucleo side phosphorylases is used for detection of mycoplasma in cell cultures.19 It is highly potent and toxic to nonproliferating and proliferating tumor cells. Recently, the use of cytotoxic bases liberated by purine nucleoside phosphorylases such as 6-methylpurine was proposed as a novel principle in the gene therapy of cancer.20... 

The lethal effect of mycoplasmas on cell cultures can be accentuated by culture in the presence of 6-methylpurine deoxyribonucleoside (6-MPDR) and this forms the basis of the MycoTect kit available from Gibco. All mycoplasmas possess high levels of adenosine phosphorylase which converts the non-toxic 6-MPDR into 6-methylpurine and 6-methylpurine ribonucleoside, both of which are toxic to mammalian cells (McGarrity and Carson, 1982). 

Few detailed studies have been done on the purine salvage enzymes of procyclic African trypanosomes. Tb. gambiense has high levels of guanine deaminase and lacks adenine and adenosine deaminase activities (8). Tb. brucei, T.b. gambiense and T.b. rhodesiense convert allopurinol into aminopyrazolopyrimidine nucleotides and incorporates these into RNA (49). This indicates that HPRTase, succino-AMP synthetase, and succino-AMP lyase are present. At least three nucleoside cleavage activities are present (Berens, unpublished results) two are hydrolases, of which one is specific for purine ribonucleosides and the other is specific for purine deoxyribonucleosides. The third nucleoside cleavage activity is a methylthioadenosine/adenosine phosphorylase. The adenosine kinase is similar to that of L. donovani (Berens, unpublished results). 

The enzymatic synthesis of ribonucleosides and 2-deoxyribonucleosides of IcPs is of a special interest. Krenitsky et al. performed direct IcP ribosylation by treating the 4-substituted base with the nucleoside uridine in the presence of uridine phosphory-lase (UPH), purine nucleoside phosphorylase (PNP), and potassium hydrophosphates at pH close to 7 (284 285). 

N. and deoxynucleosides can be synthesized via a Salvage pathway (see). TTiey are also produced by hydrolysis of nucleic acids and nucleotides. Nucleoside phosphorylases and deoxynucleoside phosphorylases catalyse the reversible, phosphate-dependent cleavage of N. and deoxyribonucleosides, forming ribose 1-phosphate or deoxyribose 1-phosphate and the free base. N. and deoxyribonucleosides can be converted into their corresponding nucleotides by the action of specific kinases. 

Deoxyribonucleoside phosphorolysis is catalyzed by purine nucleoside phosphorylase, thymidine phosphorylase, and to some extent by uridine phosphorylase the specificity of animal uridine phosphorylases differs with the cells of origin (see Chapter 12). 

The purine nucleoside phosphorylase activity of animal tissues cleaves both ribo- and deoxyribonucleosides of 6-oxypurines. For example, the the purine nucleoside phosphorylase of human erythrocytes has been highly purified and crystallized by Parks and co-workers 2) this enzyme will cleave the ribo- and deoxyribonucleosides of guanine and hypoxan-thine. Zimmerman et al. (3) have shown that purine nucleoside phosphorylase of several animal tissues has a low intrinsic activity toward adenine in the presence of ribose 1-phosphate. The cleavage of deoxyadenosine by highly purified preparations of the animal enzyme has not been reported but by analogy with adenosine, one might expect it also to be a poor substrate. The specificities of the purine nucleoside phosphorylases of E. coli and S. typhimurium differ from that of the animal enzyme in that adenosine and deoxyadenosine are readily phosphorolyzed H-6). 

Although uridine is the preferred substrate of the uridine phosphorylase of animal tissues, deoxyuridine and thymidine are also cleaved at appreciable rates. The uridine phosphorylase activity of dog tissues readily cleaves deoxyuridine and thymidine and, in fact, may be the only means by which the pyrimidine deoxyribonucleosides are phosphorolyzed in dog tissues, because thymidine phosphorylase is absent from a number of them (9) (see Chapter 12). Uridine phosphorylase of E. coli is highly specific toward the ribosyl portion of its substrate and cleavage of deoxyribonucleosides is slower relative to uridine than with the animal enzymes (5). 

Fig. 14-1. Catabolism of deoxyribonucleosides (1) cytidine deaminase (2) adenosine deaminase (3) thymidine phosphotylase (4) purine nucleoside phosphorylase (5) phosphoribomutase (6) deoxyriboaldolase.

The reversibihty of deoxyribonucleoside phosphorolysis suggests that bases might be elevated to the deoxyribonucleotide level by successive actions of a phosphorylase and a kinase ... 

The first step, deoxyribonucleoside synthesis, has been demonstrated in vitro with purified preparations of purine ribonucleoside phosphorylase and thymidine phosphorylase (11-13). However, it is uncertain whether a significant incorporation of base occurs in cells by this route, without an extracellular source of deoxyribosyl groups (see below). The possibility that deoxyribose 1-phosphate may be generated endogenously by way of deoxyriboaldolase is discussed in Section IV. 

As noted above, uridine phosphorylases of some animal tissues have an appreciable activity toward deoxyribonucleosides (9) and, accordingly, may participate in transfer reactions of this sort. 

This enzyme has not been highly purified and there is some evidence that there may be two or more enzymes involved, mth one being specific for purine-purine transfers. Specificity for deoxyribonucleosides has been shown clearly. It is noteworthy that cytosine and deoxycytidine are substrates for the frares-A-deoxyribosylase this is convincing evidence of an identity separate from the phosphorylases because deoxycytidine is not a substrate for the latter. 

Thymidine phosphorylase can also use deoxyuridine as substrate [161-163], and the purine nucleoside enzyme can use either the ribonu-cleoside or the deoxyribonucleoside forms of adenine or guanine [115,164], Uridine phosphorylase (EC 2.4.2.3) is a separate entity and will not be considered here, since its regulation is not clearly understood. The four enzymes under consideration are interrelated in function and operate in concert in the regulation of nucleoside catabolism. The mechanisms of their regulation evolved from a number of independent and seemingly devious observations and events, the essence of which may be summarized as follows. 

The poor incorporation of thymidine by prototrophic bacteria may be enhanced by methods which prevent its breakdown, either by a mutational loss of thymidine phosphorylase [162] or by the addition of other deoxyribonucleosides [178-180]. The latter also enhance the incorporation of thymine by supplying the required deoxyribose-l-P [178-181]. 

The mechanism of formation of thymine and hydroxymethylcytosine are not yet known, but the addition of a carbon to position 5 appears to involve a transfer from a folic acid derivative. As in the case of the purines, the relation between ribose and deox3nibose nucleotides is obscure. Deoxyribonucleosides can be formed from deoxyribose-l-phosphate by a nucleoside phosphorylase reaction. 

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