Nucleoside phosphorylase action

The susceptibilities of some of these fluorinated purine nucleosides to the action of enzymes are now described. In contrast to the inertness of the 2 -deoxy-2 -fluoro- and 3 -deoxy-3 -fluorocytidine analogs 739, 744, and 821 towards cytidine deaminase, the adenosine compounds 867, 883, and 906 are readily deaminated - by the adenosine deaminase in erythrocytes and calf intestine, but the resulting (deaminated) inosine compounds (from 867 and 883), as well as 888, are highly resistant - to cleavage by purine nucleoside phosphorylase (to give hypoxanthine base for the first two). The reason was discussed. Both 867 and 883 can form the 5 -triphosphates, without deamination, in human erythrocytes or murine sarcoma cells in the presence of 2 -deoxycoformycin, an adenosine deaminase inhibitor, but... 

Gilbertsen RB, Scott ME, Dong MK, Kossarek LM, Bennett MK, Schrier DJ, Sircar JC. Preliminary report on 8-amino-9-(2-thienylmethyl) guanine (PD 119,229), a novel and potent purine nucleoside phosphorylase inhibitor. Agents and Actions 1987 21 272-4. 

Arsenate will replace phosphate in all phospho-rolytic reactions, e.g., in the cleavage of glycogen by glycogen phosphorylase, of sucrose by sucrose phosphorylase, and in the action of purine nucleoside phosphorylase.b Glucose 1-arsenate or ribose-1-arsenate is presumably a transient intermediate which is hydrolyzed to glucose. The overall process is called arsenolysis. Another reaction in which arsenate can replace phosphate is the oxidation of glyceraldehyde 3-phosphate in the presence of P to form 1,3-bisphosphoglycerate ... 

A close look at this reaction reveals that in the opposite direction, the reaction is of the phosphorolysis type. For this reason, the enzymes catalyzing the reaction with ribose-l-phosphate are called phosphorylases, and they also participate in nucleic acid degradation pathways. Purine nucleoside phosphorylases thus convert hypoxanthine and guanine to either inosine and guanosine if ribose-l-phosphate is the substrate or to deoxyinosine and deoxyguanosine if deoxyribose-1-phosphate is the substrate. Uridine phosphorylase converts uracil to uridine in the presence of ribose-l-phosphate, and thymidine is formed from thymine and deoxyribose-l-phosphate through the action of thymidine phosphorylase. 

Catabolism of the nucleotides (Figure 24-3, B) begins with removal of their ribose-linked phosphate, a process catalyzed by purine 5 -nucleotidase. Removal of the ribose moiety of inosine and guanosine by the action of purine-nucleoside phosphorylase forms hypoxanthine and guanine, both of which are converted to xanthme. Xanthine is converted to uric acid through the action of xanthine oxidase. 

Purine nucleosides are cleaved by the action of purine nucleoside phosphorylase with the liberation of ribose 1-phosphate (Kl, PI). The enzyme is apparently specific for purines. The material from erythrocytes catalyzes the phosphorolysis of purine but not pyrimidine nucleosides (T6.) Purine phosphorylase activity is found widespread in nature and in many animal tissues (FIO). Friedkin and Kalckar investigated an enzyme capable of cleaving purine deoxynucleosides to the aglycone and deoxy-ribose 1-phosphate. They concluded that the enzyme was identical to that which splits purine ribonucleosides (F8, F9). This enzyme is capable of degrading inosine, xanthosine, and guanosine to forms readily attacked by other enzymes. In so doing, it permits living cells to retain the ribose and deoxyribose moieties. 

Doxifluridine (5 -DFUR, 199) is an oral prodmg of 5FU (177). Compound 199, designed to circumvent the rapid degradation of 177 by dihydropyrimidine dehydrogenase in the gut wall, is converted into 177 by action of pyrimidine nucleoside phosphorylase. 

An economically viable alternative to the synthesis of deoxyribonuclosides has been developed as a two stage process involving 2-deoxy-D-ribose 5-phosphate aldolase (DERA) (Fig. 6.5.14) (Tischer et al. 2001). The first step was the aldol addition of G3P to acetaldehyde catalyzed by DERA. G3P was generated in situ by a reverse action of EruA on L-fructose-1,6-diphosphate and triose phosphate isomerase which transformed the DHAP released into G3P. In a second stage, the action of pentose-phosphate mutase (PPM) and purine nucleoside phosphorylase (PNP), in the presence of adenine furnished the desired product. The released phosphate was consumed by sucrose phosphorylase (SP) that converts sucrose to fructose-1-phosphate, shifting the unfavorable equilibrium position of the later reaction. 

The term carbocyclic nucleoside [181] is used to describe a group of compounds structurally related to nucleosides in which the furanose ring has been replaced by a cyclopentane ring. A consequence of this substitution is an enhancement of the metabolic stability of the carbocyclic nucleosides, which are not subjected to the action of nucleoside phosphorylases and hydrolases that cleave normal nucleosides. However, from the conformational point of view the tetrahydro-furan and cyclopentane rings are similar. Thus, carbocyclic nucleosides may act as substrates or inhibitors of the enzymes that activate (kinases) and transform nucleosides and nucleotides in living cells and incorporate them into DNA. Most approaches to the synthesis of carbocyclic nucleosides begin with the construction of the nucleic acid base from a functionalized cyclopentylamine, which with some exceptions is obtained as a racemic mixture. The medicinal chemistry of carbocyclic nucleosides, as well as the synthesis of the intermediate cyclopentylamines have been reviewed [181]. This section deals only with work published after that review, directly related with anti-AIDS research. 

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. 

For example, intact Ehrlich ascites tumor cells, or extracts therefrom, transfer the ribosyl group of uridine to hypoxanthine and thereby catalyze the net synthesis of inosine this reaction depends upon the coupled actions of uridine phosphorylase and purine nucleoside phosphorylase (89). Similar ribosyl transfers have been demonstrated with bacterial cells and extracts. Krenitsky has studied the kinetics of exchange between uracil-2- C and nonisotopic uridine catalyzed by highly purified uridine phosphorylase (30) ... 

However, for adenine, guanine, and uracil, the dominant route of anabolism is by way of their ribonucleotide derivatives and traffic along the deoxyribosidic route is not ordinarily significant. Because cytosine is not a substrate for nucleoside phosphorylases, incorporation by the phos-phorylase-kinase route is not possible for this base. The other pyrimidine base of DNA, thymine, is poorly anabolized by both animal and bacterial cells, in spite of the fact that most cells possess thymidine phosphorylase, the action of which is readily reversible. This suggests that ordinarily cellular supplies of deoxyribose 1-phosphate are not available for base anabolism. Experiments are cited below in which it was demonstrated that a significant contribution to the biogenesis of deoxyribose of DNA in E. colt cells did not occur by a route other than ribonucleotide reduction. 

A means of converting free bases into deoxyribonucleotides would appear to be afforded by the sequential action of nucleoside phosphorylases and kinases however, cells have only a very limited capability for the endogenous formation of the deoxyribose 1-phosphate needed for this... 

Purine-pyrimidine deoxyribosyl transfer reactions result when reactions (1) and (2) are catalyzed by the joint actions of purine nucleoside phosphorylase and thymidine phosphorylase, that is, when the activities of these enzymes are coupled. For example, the following reaction is catalyzed by extracts of human leukocytes (18) ... 

Nucleotides are formed directly from the aglycone by a phosphori-bosyl transfer involving PRPP (Reaction 1), or from the nucleoside by the phosphorylating action of a kinase (Reaction 2). Nucleosides may also be split by phosphorylases (Reaction 3) to yield the free base which may be recovered by Reaction 1, and ribose (or deoxyribose)- -phosphate which may be further metabolized as a carbon source. The nucleoside phosphorylases are primarily catabolic in function and, though reversible, they play little, if any, role in the normal utilization of free bases. The reversibility is limited by the availability of pentose phosphates and operates only under special conditions that allow accumulation of the pentose phosphates. 

Ribose derivatives. a-o-Ribofuranose i-phosphate was first obtained by the action of nucleoside phosphorylase on nucleosides . It has been prepared by treatment of 5-O-acetyl-D-ribofuranosyl i-bromide 2,3-cyclic carbonate with triethyl ammonium dibenzyl phosphate followed by removal of the protecting groups . 

Deoxy-D-ribose 5-phosphate has been obtained by the action of a nucleoside phosphorylase and a mutase on hypoxanthine , by enzymic condensation of triose phosphate and acetaldehyde and by enzymic phosphorylation of D-2-deoxyribose . Acid hydrolysis of deoxy-adenylic or deoxy-guanylic acid yields deoxyribose 5-phosphate . A chemical s)mthesis is available . 

In liver, specific pyrophosphorylases catalyse the synthesis of nucleotides from free bases and 5-phos-phoribosyl 1-pyrophosphate alternatively, nucleosides may be formed first from bases and ribose 1-phosphate by the action of a nucleoside phosphoiy-lase (Fig.). Deoxynucleosides can be formed from bases and deoxyribose 1-phosphate by the action of deoxynucleoside phosphorylase. 

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