Rabbit erythrocyte purine nucleoside phosphorylase. Differential-inactivation studies.

1. Qualitative studies on the stability of rabbit erythrocyte purine nucleoside phosphorylase showed a marked decrease in the susceptibility of the enzyme to thermal inactivation and digestion by proteinases of different specificities in response to certain of its substrates. 2. The extent to which inosine stabilizes the enzyme against thermal and proteolytic inactivation is related in a quantitative manner to the concentration of this substrate; it is proposed that differences in the rates of inactivation of the enzyme may reflect substrate-induced conformational changes in the enzyme structure that could alter the binding properties of the enzyme in a kinetically significant way. 3. A synergistic effect in the stabilization of the enzyme is observed in response to both substrates, inosine and phosphate, when the enzyme is inactivated with Pronase. 4. In the presence of substrate an increased rate of inactivation after reaction with 5,5'-dithiobis-(2-nitrobenzoic acid) is reported. 5. Differential-inactivation studies were also carried out with calf spleen purine nucleoside phosphorylase, and the results are discussed in relation to the kinetic properties displayed by this enzyme.     

Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12.

A xanthosine-inducible enzyme, inosine-guanosine phosphorylase, has been partially purified from a strain of Escherichia coli K-12 lacking the deo-encoded purine nucleoside phosphorylase. Inosine-guanosine phosphorylase had a particle weight of 180 kilodaltons and was rapidly inactivated by p-chloromercuriphenylsulfonic acid (p-CMB). The enzyme was not protected from inactivation by inosine (Ino), 2'-deoxyinosine (dIno), hypoxanthine (Hyp), Pi, or alpha-D-ribose-1-phosphate (Rib-1-P). Incubating the inactive enzyme with dithiothreitol restored the catalytic activity. Reaction with p-CMB did not affect the particle weight. Inosine-guanosine phosphorylase was more sensitive to thermal inactivation than purine nucleoside phosphorylase. The half-life determined at 45 degrees C between pH 5 and 8 was 5 to 9 min. Phosphate (20 mM) stabilized the enzyme to thermal inactivation, while Ino (1 mM), dIno (1 mM), xanthosine (Xao) (1 mM), Rib-1-P (2 mM), or Hyp (0.05 mM) had no effect. However, Hyp at 1 mM did stabilize the enzyme. In addition, the combination of Pi (20 mM) and Hyp (0.05 mM) stabilized this enzyme to a greater extent than did Pi alone. Apparent activation energies of 11.5 kcal/mol and 7.9 kcal/mol were determined in the phosphorolytic and synthetic direction, respectively. The pH dependence of Ino cleavage or synthesis did not vary between 6 and 8. The substrate specificity, listed in decreasing order of efficiency (V/Km), was: 2'-deoxyguanosine, dIno, guanosine, Xao, Ino, 5'-dIno, and 2',3'-dideoxyinosine. Inosine-guanosine phosphorylase differed from the deo operon-encoded purine nucleoside phosphorylase in that neither adenosine, 2'-deoxyadenosine, nor hypoxanthine arabinoside were substrates or potent inhibitors. Moreover, the E. coli inosine-guanosine phosphorylase was antigenically distinct from the purine nucleoside phosphorylase since it did not react with any of 14 monoclonal antisera or a polyvalent antiserum raised against deo-encoded purine nucleoside phosphorylase.     

Synthesis of 9-(3,4-dioxopentyl)hypoxanthine, the first arginine-directed purine derivative: an irreversible inactivator for purine nucleoside phosphorylase

The synthesis of two potential arginine-directed purine-based analogues, 6-chloro-9-(3,4-dioxopentyl)purine (6) and 9-(3,4-dioxopentyl)hypoxanthine (7), is reported. Compound 7 was extensively tested as a potential affinity label of purine nucleoside phosphorylase (EC 2.4.2.1) from human erythrocytes. Evidence that 7 reacted with the catalytic center of purine nucleoside phosphorylase includes the following: (1) time-dependent inactivation of the enzyme by 7 was observed; (2) a plot of the pseudo-first-order rate constant for inactivation of the enzyme vs. concentration of 7 was hyperbolic, characteristic of saturation phenomenon; (3) substrates (Pi, arsenate, inosine) and a competitive inhibitor (formycin B) protected the enzyme from inactivation by 7. Compound 7 was 25 times more effective in inhibiting purine nucleoside phosphorylase than butanedione. Evidence that 7 modified arginine(s) includes the following: (1) when the inactivation was performed in borate, both the rate and the extent of inactivation were enhanced compared to those of the controls run in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer; (2) dialysis of inactivator reversed the inactivation in Tris-HCl but not in borate buffer. All the above evidence combined with the previous demonstration [Jordan, F., & Wu, A. (1978) Arch. Biochem. Biophys. 190, 699-704] that butanedione modified only arginines in purine nucleoside phosphorylases and the results presented here demonstrating the similarities in the behavior of butanedione and 7 imply that compound 7 can be called an arginine-directed affinity label for purine nucleoside phosphorylase.     

Blood glycoprotein from antarctic fish. Possible conformational origin of antifreeze activity

Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared to be a tetramer of molecular weight 95 000. The other enzyme, adenosine phosphorylase, was specific for adenosine and deoxyadenosine. The molecular weight of the native enzyme was 153 000 +/- 10% and the molecular weight of the subunits was 25 500 +/- 5%. This indicates a hexameric structure. The adenosine phosphorylase was inactivated by 10(-3) M p-chloromercuribenzoate and protected against this inactivation by phosphate, adenosine and ribose 1-phosphate.     

Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.     

Expression of human malaria parasite purine nucleoside phosphorylase in host enzyme-deficient erythrocyte culture. Enzyme characterization and identification of novel inhibitors

The intraerythrocytic human malaria parasite, Plasmodium falciparum, requires a source of hypoxanthine for nucleic acid synthesis and energy metabolism. Adenosine has been implicated as a major source for intraerythrocytic hypoxanthine production via deamination and phosphorolysis, utilizing adenosine deaminase and purine nucleoside phosphorylase, respectively. To study the expression and characteristics of human malaria purine nucleoside phosphorylase, P. falciparum was successfully cultured in purine nucleoside phosphorylase-deficient human erythrocytes to an 8% parasitemia level. Purine nucleoside phosphorylase activity was undetectable in the uninfected enzyme-deficient host red cells but after parasite infection rose to 1.5% of normal erythrocyte levels. The parasite purine nucleoside phosphorylase was not cross-reactive with antibody against human enzyme, exhibited a calculated native molecular weight of 147,000, and showed a single major electrophoretic form of pI 5.4 and substrate specificity for inosine, guanosine and deoxyguanosine but not xanthosine or adenosine. The Km values for substrates, inosine and guanosine, were 4-fold lower than that for the human erythrocyte enzyme. In these studies we have identified two novel potent inhibitors of both human erythrocyte and parasite purine nucleoside phosphorylase, 8-amino-5'-deoxy-5'-chloroguanosine and 8-amino-9-benzylguanine. These enzyme inhibitors may have some antimalarial potential by limiting hypoxanthine production in the parasite-infected erythrocyte.     

Hexamere purine nucleoside phosphorylase from Escherichia coli K-12. Kinetic analysis and mechanism of reaction

A kinetic analysis of the phosphorolytic reaction catalyzed by hexameric purine nucleoside phosphorylase II from E. coli K-12 in the presence and absence of reaction products was carried out. The results of the kinetic analysis are consistent with a rapid equilibrium random Bi-Bi mechanism, in which a dead-end ternary (enzyme.purine base.phosphate) complex is formed.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.     

Inactivation of purine nucleoside phosphorylase by modification of arginine residues.

Treatment of purine nucleoside phosphorylase (EC 2.4.2.1), from either calf spleen or human erythrocytes, with 2,3-butanedione in borate buffer or with phenylglyoxal in Tris buffer markedly decreased the enzyme activity. At pH 8.0 in 60 min, 95% of the catalytic activity was destroyed upon treatment with 33 mM phenylglyoxal and 62% of the activity was lost with 33 mm 2,3-butanedione. Inorganic phosphate, ribose-1-phosphate, arsenate, and inosine when added prior to chemical modification all afforded protection from inactivation. No apparent decrease in enzyme catalytic activity was observed upon treatment with maleic anhydride, a lysine-specific reagent. Inactivation of electrophoretically homogeneous calf-spleen purine nucleoside phosphorylase by butanedione was accompanied by loss of arginine residues and of no other amino acid residues. A statistical analysis of the inactivation data vis-à-vis the fraction of arginines modified suggested that one essential arginine residue was being modified.     

A second purine nucleoside phosphorylase in Escherichia coli K-12

Summary The presence of a second purine nucleoside phosphorylase in wild-type strains of E. coli K-12 after growth on xanthosine has been demonstrated. Like other purine nucleoside phosphorylases it is able to carry out both phosphorylosis and synthesis of purine deoxy- and ribonucleosides whilst pyrimidine nucleosides cannot act as substrates. In contrast to the well characterised purine nucleoside phosphorylase of E. coli K-12 (encoded by the deoD gene) this new enzyme could act on xanthosine and is hence called xanthosine phosphorylase. Studies of its substrate specificity showed that xanthosine phosphorylase, like the mammalian purine nucleoside phosphorylases, has no activity towards adenine and the corresponding nucleosides. Determinations of K _m and gel filtration behaviour was carried out on crude dialysed extracts. The presence of xanthosine phosphorylase enables E. coli to grow on xanthosine as carbon source. Xanthosine was the only compound found which induced xanthosine phosphorylase. No other known nucleoside catabolising enzyme was induced by xanthosine. The implications of non-linear induction kinetics of xanthosine phosphorylase is discussed.     

Chicken liver purine nucleoside phosphorylase activity dependency of the redox state of sulfhydryl groups

1. Double reciprocal plots (1/v vs 1/S) for nucleoside substrates of chicken liver purine nucleoside phosphorylase were non linear at high inosine or deoxyinosine concentrations (greater than 0.1 mM). The appearance of downward curvatures may be correlated with the oxidation of sulfhydryl groups of the enzyme. 2. 5,5'-Dithiobis-(2-nitrobenzoic acid) reacts with four sulfhydryl groups in the native enzyme, but upon denaturation with sodium dodecylsulfate six sulfhydryl groups react with this reagent. 3. Inosine, ribose-1-phosphate, hypoxanthine and orthophosphate partially protect sulfhydryl groups from the reaction with Ellman's reagent. 4. Inhibition of purine nucleoside phosphorylase by p-chloromercuribenzoate and 5,5'-dithiobis-(2-nitrobenzoic acid) follows a second order reaction kinetics.     

Deoxyguanosine toxicity in a mouse T lymphoma: relationship to purine nucleoside phosphorylase-associated immune dysfunction.

The absence of either of the enzymes adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with an immunodeficiency disease. Because all four nucleoside substrates of the enzyme purine nucleoside phosphorylase accumulate in the urine of patients who lack this enzyme (Cohen et al., 1976), we examined the toxicity of each of the four substrates using a mouse T cell lymphoma (S49) in continuous culture. Of the four substrates (inosine, deoxyinosine, guanosine and deoxyguanosine), only deoxyguanosine is cytotoxic at concentrations lower than 100 μM; furthermore, only deoxyguanosine is directly phosphorylated in S49 cells. Mutant S49 cells lacking deoxycytidine kinase (EC 2.7.1.74) are resistant to the toxic effects of deoxyguanosine, and these same mutants do not phosphorylate deoxyguanosine. Thus the cytotoxicity of exogenous deoxyguanosine correlates with the intracellular concentration of accumulated deoxyGTP.The addition of deoxyguanosine results in the depletion of deoxyCTP in S49 cells, indicating that deoxyGTP is an inhibitor of ribonucleotide reductase. Furthermore, the addition of deoxycytidine prevents the toxic effects of deoxyguanosine. Thus a therapy for purine nucleoside phosphorylase-deficient patients might include deoxycytidine to alleviate the proposed deoxyCTP starvation in those tissues capable of phosphorylating deoxyguanosine.     

Purification and characterization of human erythrocyte purine nucleoside phosphorylase and its subunits.

Purine nucleoside phosphorylase (EC 2.4.2.1; purine nucleoside:orthophosphate ribosyltransferase) from fresh human erythrocytes has been purified to homogeneity in two steps with an overall yield of 56%. The purification involves DEAE-Sephadex chromatography followed by affinity chromatography on a column of Sepharose/formycin B. This scheme is suitable for purification of the phosphorylase from as little as 0.1 ml of packed erythrocytes. The native enzyme appears to be a trimer with native molecular weight of 93,800 and the subunit molecular weight of 29,700 +/- 1,100. Two-dimensional gel electrophoresis of the purified enzyme under denaturing conditions revealed four major separable subunits (numbered 1 to 4) with the same molecular weight. The apparent isoelectric points of subunits 1 to 4 in 9.5 M urea are 6.63, 6.41, 6.29, and 6.20, respectively. The different subunits are likely the result of post-translational modification of the enzyme and provide an explanation of the complex native isoelectric focusing pattern of purine nucleoside phosphorylase from erythrocytes. Three of the four subunits are detectable in two-dimensional electrophoretic gels of crude hemolysates. Knowing the location of the subunits of purine nucleoside phosphorylase in a two-dimensional electropherogram allows one to characterize the purine nucleoside phosphorylase in crude cell extracts from individuals with variant or mutant purine nucleoside phosphorylase as demonstrated in a subsequent communication. Partial purification of the phosphorylase from 1 ml of erythrocytes on DEAE-Sephadex increases the sensitivity of detection of the subunits to the 0.3% level.     

Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism,hydrolysis of 7-methylguanosine,and oligomeric state in solution

The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 microg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 microg/ml-0.29 mg/ml). At concentrations below 0.9 microg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (m7Guo) with a catalytic rate constant 1.3x10(-3) x s(-1) as compared with the rate of 38 s(-1) for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m7Guo, 70 s(-1), is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 microM-30 mM phosphate is consistent with only one dissociation constant for phosphate, K(d)=220+/-120 microM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 microM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.     

Purine nucleoside phosphorylase. Allosteric regulation of a dissociating enzyme

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is a trimeric enzyme that readily dissociates to the monomer. Dilution of enzyme from 20 to 0.02 microgram of protein/ml is accompanied by a greater than 50-fold increase in the specific activity (vtrimer = 0.23 nmol/min/microgram; vmonomer = 12.5 nmol/min/micrograms). Gel permeation chromatography in the presence of the substrate phosphate shows the enzyme to be predominantly trimeric at 50 mM Pi and predominantly monomeric at 100 mM Pi, when experiments are done at 24 degrees C. No significant dissociation was observed at 4 degrees C with Pi or at either temperature with the substrate inosine. As measured by dissociation, the L0.5 for Pi is 88 mM and thus significantly higher than the Km of 3.1 mM for Pi. Enzyme activity as a function of phosphate concentration showed negative cooperativity, but the conformational response measured by the change in native Mr during dissociation showed positive cooperatively toward Pi. These data support a model for two separate phosphate binding sites on the enzyme. The activity and stability of purine nucleoside phosphorylase are quite sensitive to the concentration of the enzyme as well as appropriate substrates. Although the monomer is interpreted as being a fully active form of the enzyme, the data in general are most consistent with the enzyme functioning in vivo as a regulated trimer.     

Thyroid purine nucleoside phosphorylase. II. Kinetic model by alternate substrate and inhibition studies.

Nucleoside analog inhibition studies have been conducted on thyroidal purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) which catalyzed an ordered bi-bi type mechanism where the first substrate is inorganic phosphate and the last product is ribose 1-phosphate. Heterocyclic- and carbohydrate-modified nucleoside inhibitors demonstrate mixed type inhibition suggesting such analogs show an affinity (Ki) for the free enzyme. A kinetic model is proposed which supports the observed inhibition patterns. These studies together with alternate substrate studies indicate that nucleoside binding requires a functional group capable of hydrogen bonding at the 6-position of the purine ring and that the orientation of the bound substrate may be syn. Proper geometry of the phosphate is dependent upon the 3'-substituent to the orientated below the furanose ring. The 5'-hydroxyl group is required for substrate activity. The proposed rate limiting step of the phosphorylase mechanism is the enzymatic protonation of the 7-N position of the nucleoside.     

Purine nucleoside phosphorylase from bovine liver.

1. Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, E.C. 2.4.2.1) from liver of cattle, Bos taurus, was purified to homogeneity. Some properties of the enzymes from three different bovine tissues were compared and discussed. 2. The enzyme has a molecular weight of 83,000, a sedimentation coefficient of 5.3 S, a Stokes' radius of 3.71 nm, a frictional ratio of 1.30 and a subunit molecular weight of 30,000. 3. Optimal pH for xanthosine degradation is around 5.5, whereas a broad pH activity profile for inosine degradation was observed between 5.0 and 7.5. Lineweaver-Burk plots curved downward at high concentrations of substrates, inosine, phosphate and arsenate.     

Liver purine nucleoside phosphorylase in Camelus dromedarius: purification and properties

1. Purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyl transferase, EC 2.4.2.1) was purified to electrophoretic homogeneity from the liver of Camelus dromedarius. 2. The enzyme appears to be a dimer with a 44,000 subunit mol. wt and displays non-linear kinetics with concave downward curvature in double reciprocal plots with respect to both inosine and orthophosphate as variable substrates. 3. The effect of thiol compounds on the enzyme activity and of pH on kinetic parameters is reported.     

New phosphate binding sites in the crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with phosphate and formycin A

Purine nucleoside phosphorylase (PNP) from Escherichia coli is a homohexamer that catalyses the phosphorolytic cleavage of the glycosidic bond of purine nucleosides. The first crystal structure of the ternary complex of this enzyme (with a phosphate ion and formycin A), which is biased by neither the presence of an inhibitor nor sulfate as a precipitant, is presented. The structure reveals, in some active sites, an unexpected and never before observed binding site for phosphate and exhibits a stoichiometry of two phosphate molecules per enzyme subunit. Moreover, in these active sites, the phosphate and nucleoside molecules are found not to be in direct contact. Rather, they are bridged by three water molecules that occupy the "standard" phosphate binding site.     

Allosteric regulation of purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (EC 2.4.2.1) from bovine spleen is allosterically regulated. With the substrate inosine the enzyme displayed complex kinetics: positive cooperativity vs inosine when this substrate was close to physiological concentrations, negative cooperativity at inosine concentrations greater than 60 microM, and substrate inhibition at inosine greater than 1 mM. No cooperativity was observed with the alternative substrate, guanosine. The activity of purine nucleoside phosphorylase toward the substrate inosine was sensitive to the presence of reducing thiols; oxidation caused a loss of cooperativity toward inosine, as well as a 10-fold decreased affinity for inosine. The enzyme also displayed negative cooperativity toward phosphate at physiological concentrations of Pi, but oxidation had no effect on either the affinity or cooperativity toward phosphate. The importance of reduced cysteines on the enzyme is thus specific for binding of the nucleoside substrate. The enzyme was modestly inhibited by the pyrimidine nucleotides CTP (Ki = 118 microM) and UTP (Ki = 164 microM), but showed greater sensitivity to 5-phosphoribosyl-1-pyrophosphate (Ki = 5.2 microM).