Purine nucleoside phosphorylase reaction catalyzed

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. 

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. 

Kline PC, Schramm VL (1995) Pre-steady-state transition-state analysis of the hydrolytic reaction catalyzed by purine nucleoside phosphorylase. Biochemistry 34 1153-1162... 

Reactions catalyzed by adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). ADA and PNP participate in the purine catabolic pathway, and deficiency of either leads to immunodeficiency disease. [Slightly modified and reproduced, with permission, from N. M. Kredich and M. S. Hershfield, Immunodeficiency diseases caused by adenosine deaminase and purine nucleoside phosphorylase deficiency. In The Metabolic Basis of Inherited Disease, 6th ed., C. S. Scriver, A. L. Beaudet, W. S. Sly, and D. Valle, Eds. New York McGraw-Hill (1989).]... 

Purine nucleoside phosphorylase (PNP). In contrast to lU-NH, PNP appears to use extensive contacts with the purine ring to promote catalysis and relatively few contacts with the ribosyl ring. The crystal structure of PNP has been determined in a complex with an iminosugar inhibitor, immucillin H, which was developed based on TS analyses of PNP-catalyzed hydrolysis and arsenolysis reactions. TS analysis revealed that the enzyme catalyzes a dissociative A Dn mechanism. The crystal structure was compared with the structures determined by Ealick and coworkers ° of PNP in a Michaelis complex analogue, PNP-inosine-sulfate, and a product complex, PNP-a-D-ribose-l-phosphate-hypoxanthine. 

Glycogen degradation is a phospho-rolysis reaction (breaking of a bond using a phosphate ion as a nucleophile). Enzymes that catalyze phosphorolysis reactions are named phosphorylase. Because more than one type of phosphorylase exists, the substrate usually is included in the name of the enzyme, such as glycogen phosphorylase or purine nucleoside phosphorylase. 

Fig. 41.10. Salvage of bases. The purine bases hypoxanthine and gnanine react with PRPP to form the nucleotides inosine and gnanosine monophosphate, respectively. The enzyme that catalyzes the reaction is hypoxanthine-gnanine phosphoribosyltransferase (HGPRT). Adenine forms AMP in a reaction catalyzed by adenine phosphoribosyltransferase (APRT). Nucleotides are converted to nucleosides by 5 -nucleotidase. Free bases are generated from nncleosides by purine nucleoside phosphorylase. Deamination of the base adenine occurs with AMP and adenosine deaminase. Of the purines, only adenosine can be directly phosphorylated back to a nucleotide, by adenosine kinase.

For the alternative production of nucleosides, Zuffi and Monciardini have reported a process by a transglycosylation reaction catalyzed by uridine phosphor-ylase and purine nucleoside phosphorylase. Specifically, their invention generally relates to a process for immobilizing cells and to the use of a resin for immobilizing cells, and the process is exemplified by the production of 306 from l-p-o-arabino-furanosyluracil 315 and 314 (Scheme 12.82). Similar biotransformations for the preparation of 306 and 307 from 314 and arabinofuranosyluracil have been reported by Farina et al. (using Enterobacter aerogenes), and Hummel-Marquardt et al. (using esterases (e.g., pig liver esterase) or lipases). ... 

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... 

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. 

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) ... 

Purine nucleoside phosphorylase and thymidine phosphorylase have been shown to catalyze transfer of the deoxyribosyl group from one base to another by reaction sequences that involve the intermediate formation of free deoxyribose 1-phosphate by the following mechanism ... 

In purine-purine deoxyribosyl transfer, a single enzyme, purine nucleoside phosphorylase (see Chapter 10), participates in both reactions (1) and (2). For example, the purified enzyme catalyzes the following reaction (16) ... 

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) ... 

In addition to participation in the deoxyribosyl transfer reactions described above, in which free deoxyribose 1-phosphate is formed as an intermediate, thymidine phosphorylase also catalyzes deoxyribosyl transfers involving thymine and uracil in which deoxyribosyl phosphate is an intermediate but is enzyme-bound (18-20). Such transfers require non-stoichiometric amounts of phosphate (19). The reaction mechanisms of uridine phosphorylase and purine nucleoside phosphorylase are not of this type and, accordingly, direct deoxyribosyl transfers occur only between substrates for thymidine phosphorylase, as exemplified by reaction (5) above and the following (15) (the asterisk indicates C-labeling) ... 

Scheme 14.13. A representation of a salvage reaction in which a purine (adenine, A) undergoes reaction with a-D-ribose-l-phosphate in what is represented as an SN2-type process but which almost certainly involves participation by the ring oxygen as well as purine nucleoside phosphorylase (EC 2.4.2.1). NB The name of the enzyme as a phosphorylase refers to its potential to catalyze the reverse of the reaction shown.

On the other hand, as seen in this chapter and in earlier chapters, the formation of phosphates of adenine (e.g., AMP, ADP, and ATP), guanidine (e.g., GTP), cytosine (e.g.,cytidine monophosphate [CMP]), uracil (e.g., uridine monophosphate [UMP]), and dTMP have all involved the carbohydrate scaffold as a building block for the formation of the finished heterocyclic base (purine or pyrimidine). It is also important to realize that, as part of nucleotide salvage pathways, it has been found that a family of enzymes collectively known as phosphorylases serves to catalyze reactions between free bases and phosphate esters of carbohydrates (and related compounds). For example, as shown in Scheme 14.13, the generalized enzyme, purine nucleoside phosphorylase (EC 2.4.2.1), catalyzes the conversion of a purine with... 

The ability to accurately compute kinetic isotope effects (KIEs) for chemical reactions in solution and in enzymes is important because the measured KIEs provide the most direct probe to the nature of the transition state and the computational results can help rationalize experimental findings. This is illustrated by the work of Schramm and co-workers, who have used the experimental KIEs to develop transition state models for the enzymatic process catalyzed by purine nucleoside phosphorylase (PNP), which in turn were used to design picomolar inhibitors. In principle, Schramm s approach can be applied to other enzymes however, in order to establish a useful transition state model for enzymatic reactions, it is often necessary to use sophisticated computational methods to model the structure of the transition state and to match the computed KIEs with experiments. The challenge to theory is the difficulty in accurately determining the small difference in free energy of activation due to isotope replacements, especially for secondary and heavy isotope effects. Furthermore, unlike studies of reactions in the gas phase, one has to consider... 

Previously constructed recombinant strain E. coli BM-D6 produced homologous purine nucleoside phosphorylase (PurNPase) acting (like PyrNPase) as a key catalyst transforming pyrimidine nucleosides into modified purine nucleosides via enz5miatic transglycosylation reaction [7]. PurNPase catalyzes stereoselective reaction of intermediate a-D-pentofuranose-1-phosphate (product of pyrimidine nucleoside phosphorolysis mediated by PyrNPase) condensation with purine heterocyclic base leading to formation of modified purine nucleoside. 

Nucleoside phosphorylases catalyze the reversible phosphorolysis in nucleosides and the transferase reaction involving purine or pyrimidine bases [42]. Scheme... 

Much of the motivation for the study of XO electrochemistry is the development of amperometric biosensors. Commercially available bovine XO is invariably the enzyme of choice used in these applications. High levels of hypoxanthine are linked with asphyxia in newborns, SIDS and hypoxia in general. Coupled with other enzymes, such as purine nucleoside phos-phorylase, XO can be used to determine phosphate coneentrations in clinical, food and waste samples. Purine nueleoside phosphorylase catalyzes the phosphorylation of inosine liberating hypoxanthine and ribose-l-phosphate (Scheme 5.5). In the presence of XO, hypoxanthine is oxidized to xanthine and produces one equivalent of H2O2. Thus due to the stoichiometry of the reaction one equivalent of H2O2 is produced for every phosphate anion present. ... 

Amino-4-imidazole carboxamide ribotide, a precursor only two steps removed (formylation and cycli-zation) from inosinic acid, can be synthesized by the direct condensation of the imidazole with 5-phosphori-bosyl pyrophosphate. The enzyme catalyzing this reaction was purified from an acetone powder of beef liver. The same enzyme (AMP pyrophosphorylase) catalyzes the condensation of adenine, guanine, and hypoxan-thine. Nucleoside phosphorylase is an enzyme that catalyzes the formation of a ribose nucleoside from a purine base and ribose-1-phosphate. Guanine, adenine, xanthine, hypoxanthine, 2,6-diaminopurine, and aminoimidazole carboxamide are known to be converted to their respective nucleosides by such a mechanism. In the presence of a specific kinase and ATP, the nucleoside is then phosphorylated to the corresponding nucleotide. 

In the purine degradation pathway IMP and GMP are dephosphorylated by nonspecified phosphatases to the corresponding nucleosides, which can leave the bacteria either spontaneously or facilitated by an exporter, for example, PbuE [290, 291]. Further degradation of the nucleosides to the nucleobases is catalyzed by the nucleotide phosphorylases PunA and DeoD [292]. The bases can be salvaged by Hpt-catalyzed reactions with PRPP to IMP and GMP. 

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