Enzyme purine nucleoside phosphorylase

X-ray crystal structures of the beef purine nucleoside phosphorylase enzyme with a substrate-analogue complex (inosine and the non-nucleophilic sulfate), a transition state-analogue complex (immucillin H and phosphate) and a product complex (inosine and ribose-l-phosphate) led to the conclusion that most atomic motion was in the anomeric centre, which swung from attachment to... 

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... 

The review articles by Schramm (1998, 2003) provide a number of examples of the successful application of this protocol to the design of enzyme-specific transition state-like inhibitors. Among these, the transition state inhibitors of human purine nucleoside phosphorylase (PNP) are particularly interesting from a medicinal chemistry perspective, as examples of these compounds have entered human clinical trials for the treatment of T-cell cancers and autoimmune disorders. 

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 ... 

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of E. coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, FA. The results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme <2004MI377>. 

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 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... 

Melki, R., Fievez, S., and Carlier, M.-F. (1996). Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mer-capto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay. Biochemistry 35, 12038-12045. 

Position of Bond Cleavage PNP (EC 2.U.2.1) from human erythrocytes (homogeneous, purified by formycin B affinity chromatography) as well as from E, coli were allowed to equilibrate a mixture of R-l-[1 0lj]-P, pl Oij, hypoxanthine and inosine at pH 7-00 in 10 mm NMR tubes. The chemical shift differences of the 31p nuclei of the two R-l-P s (13 9 Hz for the human erythrocytic and 13.1 Hz for the E. coli enzyme) as well as of the two P3 resonances (13 9 Hz for erythrocytic and 13-7 Hz for E. coli source) clearly indicated C-0 bond cleavage by these enzymes as well. In addition, no evidence was found over the time course of the NMR measurements (l hr) for purine nucleoside phosphorylase catalyzed exchange of pl o + H2O (solvent) J randomized P. Therefore,... 

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. 

Finally, the serum sample was incubated with the enzyme purine nucleoside phosphorylase and rechromatographed (Fig. 14C). From the disappearance of the inosine peak and the appearance of a peak with the retention time of hypoxanthine, it can be concluded that the peak under investigation was indeed inosine. 

The most recent application of RPLC to the analysis of enzymes has been reported by Halfpenny and Brown (HI). An assay for purine nucleoside phosphorylase, a key mediator in the purine salvage pathway, has been developed and optimal conditions for the analysis determined. Figure 20 illustrates the simultaneous separation of the substrate, inosine, and products, uric acid and hypoxanthine. In another analysis. Halfpenny and Brown (H2) developed an assay for hypoxanthine-guanine phos-phoribosyltransferase. Deficiency of this enzyme has been associated with Lesch-Nyhan syndrome as well as primary gout. The activity of the enzyme is determined by measurement of the decrease of the substrate, hypoxanthine, and increase in the product, inosine-5 -monophosphoric acid. A major advantage of using HPLC for enzyme assays is that the simultaneous measurement of both substrate and product reduces the error due to interference from competing enzymes. 

The enzyme, purine nucleoside phosphorylase (PNP), is directly involved with blood levels of T-cells. Low levels of this enzyme will inhibit T-ceU prohferation. Drugs that inhibit the enzyme can also be expected to act against proliferation of malignant T-cells. The PNP inhibitor forodesine (36) has shown early activity against T-cell mahgnancies. Treatment of the deazapurine (32) with lithium leads to derivative 33... 

The XLL cross-axis CPC, with a 250-mL capacity column, was used for the purification of recombinant enzymes such as purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UrdPase) from a crude Escherichia coli lysate. The polymer-phase system used in these separations was 16% (w/w) PEG 1000-12.5% (w/w) potassium phosphate at pH 6.8. The separation was performed at 750 rpm at a flow rate of 0.5 mL/min using the upper phase as a mobile phase. About 1.0 mL of crude lysate, containing PNP in 10 mL of the above solvent system, was loaded into the multilayer coil. Purified PNP was harvested in 45-mL fractions. The SDS-PAGE analysis clearly demonstrated that PNP was highly purified in a one-step elution with the XLL cross-axis CPC. 

In a commercially available assay, serum NTP catalyzes the hydrolysis of IMP to yield inosine, which is then converted to hypoxanthine by purine-nucleoside phosphorylase (EC 2.4.2.1). Hypoxanthine is oxidized to urate with xanthine oxidase (EC 1.2.3.2). Two moles of hydrogen peroxide are produced for each mole of hypoxanthine liberated and converted to uric acid. The formation rate of hydrogen peroxide is monitored by a spectrophotometer at 510nm by the oxidation of a chromogenic system. The effect of ALPs on IMP is inhibited by p-glycerophosphate. This material is substrate for ALP but not for NTP, and by forming substrate complexes with the former enzyme, it reduces the proportion of the total ALP activity that is directed to the hydrolysis of the NTP substrate, IMP. ... 

Other studies have examined the association between the activity of TPMT and other enzymes in the purine pathway and AZA toxicity. In one study, TPMT, HPRT, 5 -nucleotidase, and purine nucleoside phosphorylase activity in the red blood cells (RBC) of 33 RA patients on AZA (dose of approximately 2 mg/ kg/day) and 66 controls was measured. Fourteen RA patients with low (p = 0.004) and seven patients with intermediate TPMT activity (RR 3.1) developed AZA toxicity when compared to patients with normal TPMT activity [66]. Another study measured TPMT activity in 3 RA patients who had experienced AZA-induced hematologic toxicity and 16 RA patients without AZA toxicity. Two patients with AZA-induced hematologic toxicity were TPMT... 

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. 

T6. Tsuboi, K. K., and Hudson, P. B., Enzymes of the human erythrocyte. I. Purine nucleoside phosphorylase Isolation procedure. J. Biol. Chem. 224, 879-887 (1957). 

Mincer, J.S., Nunez, S. and Schwartz, S.D. (2004). Coupling protein dynamics to reaction center electron density in enzymes an electronic protein promoting vibration in human purine nucleoside phosphorylase. J. Theor. Comp. Chem. 3, 501-509... 

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