Nucleoside phosphorylase, function

Adenosine deaminase deficiency is associated with an immunodeficiency disease in which both thymus-derived lymphocytes (T cells) and bone marrow-derived lymphocytes (B cells) are sparse and dysfunctional. Purine nucleoside phosphorylase deficiency is associated with a severe deficiency of T cells but apparently normal B cell function. Immune dysfunctions appear to result from accumulation of dGTP and dATP, which inhibit ribonucleotide reductase and thereby deplete cells of DNA precursors. 

This rational approach to drug design has been adopted in developing a specific inhibitor of the human cellular enzyme, purine nucleoside phosphorylase (PNP). PNP functions in the purine salvage pathway, catalysing the reversible reaction shown below ... 

The enzyme has been isolated from both eukaryotic and prokaryotic organisms [2] and functions in the purine salvage pathway [1,3]. Purine nucleoside phosphorylase isolated from human erythrocytes is specific for the 6-oxypurines and many of their analogs [4] while PNPs from other organisms vary in their specificity [5]. The human enzyme is a trimer with identical subunits and a total molecular mass of about 97,000 daltons [6,7]. Each subunit contains 289 amino acid residues. 

The conversions of inosine to hypoxanthine (Fig. 25-17, step e), of guanosine to guanine (step g), and of other purine ribonucleosides and deoxyribonucleo-sides to free purine bases are catalyzed by purine nucleoside phosphorylase.318 321b Absence of this enzyme also causes a severe immune deficiency which involves the T cells. However, B cell function is not impaired.312 315 322... 

Shi W, Ting LM, Kicska GA, Lewandowicz A, Tyler PC, Evans GB, Furneaux RH, Kim K, Almo SC, Schramm VL (2004) Plasmodium falciparum purine nucleoside phosphorylase crystal structures, immucillin inhibitors, and dual catalytic function. J. Biol. Chem. 279 18103-18106... 

Yokomatsu, T.. Hayakawa. Y.. Suemune, K., Kihara, T., Soeda, S., Shimeno, H., and Shibuya, S., Synthesis and biological evaluation of 1, l-difluoro-2-(tetrahydro-3-furanyl)ethylphosphonic acids possessing a N -purinylmethyl functional group at the ring. A new class of inhibitors for purine nucleoside phosphorylases. Bioorg. Med. Chem. Lett., 9, 2833, 1999. 

Erion, M.D., Takabayashi, K., Smith, H., Kessi, J., Wagner, S., Honger, S., Shames, S. and Ealick, S.E. (1997). Purine nucleoside phosphorylase. 1. Structure-function studies. 

I. Reactions of Hypoxanthine. The fact that hypoxanthine is an active intermediate in normal cells directs attention to the three chemical reactions hypoxanthine can undergo in the mammal (Fig. 2). It can be converted to inosine by reaction of the purine with ribose 1-phosphate catalyzed by purine nucleoside phosphorylase. This reaction is probably primarily a phosphorolytic reaction, in vivo, and converts inosine to hypoxanthine and probably does not function to convert hypoxanthine to inosine. There does exist a limited concentration of... 

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. 

In 1975, Giblett and co-workers (18) described a deficiency of the enzyme purine nucleoside phosphorylase in patients with severe combined immunodeficiency disease characterized clinically by a severe abnormality of T-lymphocyte function. ile these patients often exhibit hypouricemia as a result of the deficiency of purine nucleoside phosphorylase, they exhibit at the same time accelerated levels of purine biosynthesis de novo with increased excretion of adenosine, deoxyinosine,guanosine, and deoxyguanosine (19). In addition, the intracellular levels of PRPP are elevated in patients with purine nucleoside phosphorylase deficiency. It is assumed, therefore, that the accelerated rate of purine biosynthesis may be due, at least in part, to the elevated levels of PRPP. It has been suggested that a decreased availability of the substrates hypoxanthine and guanine leads to a decreased functional activity of the enzyme HPRT thus leading to decreased consumption of PRPP and hence the elevated levels observed. 

Although purine nucleoside phosphorylase is known to function in the... 

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. 

K.C.Rich,E.Mejians,I.H.Fox. Purine nucleoside phosphorylase deficiency improved metabolic and immunological function with erythrocyte transfusion. N.Eng.J.Med.303 973 (1980). 

Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency have been recognized as the primary cause of an associated immune deficiency syndrome. A number of mechanisms have been proposed to explain the predominant effect of these enzyme deficiencies on the development and function of the lymphoid system. One of the mechanisms concerns the phosphorylation of accumulated metabolic compounds i.e. deoxyadenosine (dAdo) in case of ADA-deficiency and deoxyguanosine (dGuo) in case of PNP deficiency in the lymphoid cells and particularly in thymocytes (1). Indeed increased deoxyATP and deoxyGTP levels have been found in the lymphocytes of ADA- and PNP-deficient patients respectively (2,3). These triphosphates may inhibit the enzyme ribonucleotide reductase which leads to a depletion of deoxyCTP and interference with lymphocytic DNA-synthesis (1). 

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. 

To demonstrate polymerase activity in a model cell, Chakrabarti et al. [79] encapsulated polynucleotide phosphorylase in vesicles composed of dimyris-toylphosphatidylcholine (DMPC). This enzyme can produce RNA from nucleoside diphosphates such as adenosine diphosphate (ADP) and does not require a template, so it has proven useful for initial studies of encapsulated polymerase activity (Fig. 10a). Furthermore, DMPC liposomes are sufficiently permeable so that 5-10 ADP molecules per second enter each vesicle. Under these conditions, measurable amounts of RNA in the form of polyadenylic acid were synthesized and accumulated in the vesicles after several days incubation. The enzyme-catalyzed reaction could be carried out in the presence of a protease external to the membrane, demonstrating that the vesicle membrane protected the encapsulated enzyme from hydrolytic degradation. Similar behavior has been observed with monocarboxylic acid vesicles [80], and it follows that complex phospholipids are not required for an encapsulated polymerase system to function. 

The phosphorylase can catalyze the formation of inosine or deoxyinosine, and of guanosine or deoxyguano-sine, but not adenosine or deoxyadenosine. However, the last two nucleosides can be converted to inosine and deoxyinosine by adenosine deaminase. The normal function of the phosphorylase appears to be the formation... 

Generally, all conversions in the biosynthetic direction, i.e. iPARMP— iPAR— iPA (catalysed by 5 -nucleotidase, (EC 3.1.3.5), and adenosine nucleosidase, (EC 3.2.2.7), respectively, c/. Fig. 2) may also proceed in the opposite direction, i.e. base-— nucleoside — nucleotide (catalysed by adenosine phosphorylase and adenosine kinase, respectively). All these enzymes require both Ade and iPA or Ado and iPAR, respectively, as substrates. They were characterised in wheat germ [15-18] and lupin seeds [19]. Interestingly, no K, -constants were reported for Z-type cytokinins (see summary in [22]). However, as seen in H-labelled Z-derivatives feeding experiments, Z-type cytokinins are also interconverted in a similar way [82,121,122]. Moreover, the specificity of these enzymes is not too strict with respect to the side chain configuration and one may speculate that this complex may function for most if not all native cytokinins [21,81]. 

The same question was studied by Niessing and Sekeris (1970) who found that after dissociation of SOS particles in 2.5 Af NaCl their protein moiety acquires the properties of a very specific RNase. When the protein was mixed with a giant D-RNA (50 to 70S) the latter underwent a limited, stepwise degradation to SOS and then to 18S molecules. The authors suggested that the RNase of the SOS particles is a phosphorylase. This conclusion was supported by the observations that orthophosphate stimulates the reaction and nucleoside diphosphates inhibit it. However, more direct evidence is needed, since knowledge of the existence of a specific RNase in informofers would be very helpful in understanding of their functions. 

Polynucleotide phosphorylase an enzyme catalysing the synthesis in vitro of polyribonucleotides. 5 -Nucleoside diphosphates are added to oligonucleotide starter molecules (primers) with the release of phosphate. The resulting sequence depends on the availability of components for the reaction. Thus it is possible to synthesize homopolymers (poly A, poly U, etc.) or Copolymers (see) (e.g. poly AU). The function of P.p. in the cell is not clear. Since it also acts as an exonuclease and catalyses the reverse reaction, it is possible that the enzyme functions in the synthesis and remobilization of storage polynucleotides. 

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. 

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