scholarly journals Rabbit erythrocyte purine nucleoside phosphorylase. Differential-inactivation studies

1979 ◽  
Vol 179 (1) ◽  
pp. 29-34 ◽  
Author(s):  
B Savage ◽  
N Spencer
Keyword(s):  
Conformational Changes ◽  
Purine Nucleoside Phosphorylase ◽  
Thermal Inactivation ◽  
Purine Nucleoside ◽  
Kinetic Properties ◽  
Rabbit Erythrocyte ◽  
Nucleoside Phosphorylase ◽  
Nitrobenzoic Acid ◽  
Quantitative Manner ◽  
The Stability

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.

scholarly journals Rabbit erythrocyte purine nucleoside phosphorylase. Initial-velocity studies

1979 ◽  
Vol 179 (1) ◽  
pp. 21-27 ◽  
Author(s):  
B Savage ◽  
N Spencer
Keyword(s):  
Initial Velocity ◽  
Purine Nucleoside Phosphorylase ◽  
Competitive Inhibition ◽  
Purine Nucleoside ◽  
Kinetic Properties ◽  
Rabbit Erythrocyte ◽  
Nucleoside Phosphorylase ◽  
Nitrobenzoic Acid ◽  
Wide Range ◽  
Partial Inactivation

1. Concave-downward double-reciprocal plots were obtained for rabbit erythrocyte purine nucleoside phosphorylase when the concentration of Pi was varied over a wide range at a fixed saturating concentration of either inosine or deoxyinosine. Similar behaviour was also displayed by the calf spleen enzyme. 2. The degree of curvature of double-reciprocal plots was greatly modified by the presence of SO42-, introduced into the assay mixture with the linking enzyme xanthine oxidase; competitive inhibition by SO42- was observed over a narrow range of high Pi concentrations. 3. Partial inactivation with 5,5′-dithiobis-(2-nitrobenzoic acid) resulted in a marked alteration in the kinetic properties of the enzyme when Pi was the variable substrate. 4. Initial-velocity data are expressed in the form of Hill plots, and the significance of such plots is discussed.

scholarly journals Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marta Narczyk ◽  
Łukasz Mioduszewski ◽  
Aleksandra Oksiejuk ◽  
Maria Winiewska-Szajewska ◽  
Beata Wielgus-Kutrowska ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Active Sites ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Kinetic Properties ◽  
Nucleoside Phosphorylase ◽  
E Coli ◽  
Site Model ◽  
Open Conformation

AbstractE. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein–ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.

Properties of Purine Nucleoside Phosphorylase (PNP) of Mammalian and Bacterial Origin

1990 ◽  
Vol 45 (1-2) ◽  
pp. 59-70 ◽  
Author(s):  
Agnieszka Bzowska ◽  
Ewa Kulikowska ◽  
David Shugar
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
Modified Nucleosides ◽  
Purine Nucleoside ◽  
Kinetic Properties ◽  
Bacterial Enzymes ◽  
Structural Requirements ◽  
Nucleoside Phosphorylase ◽  
E Coli ◽  
Purine Ring ◽  
Substrate Inhibitor

Purine nucleoside phosphorylase (PNP), from calf spleen, human erythrocytes and E. coli have been examined with regard to structural requirements of substrates and inhibitors. Kinetic parameters (Km, Vmax/Km) for a variety of N(1) and/or N(7)-methylated analogues of guanosine, inosine and adenosine have been evaluated for all three enzym es. The substrate and/or inhibitor properties of purine riboside, 1,6-dihydropurine riboside, some deazapurine nucleosides: 3-deaza- and 7-deazainosine, 1,3-dideazapurine riboside (ribobenzimidazole), and a variety of acyclonu cleosides, have been determined with mammalian and bacterial enzymes. Overall results indicate distinct similarities of kinetic properties and structural requirements of the two mammalian enzymes, although there are some differences as well. The N(1) and O6 of the purine ring are necessary for substrate-inhibitor activity and constitute a binding site for the mammalian (but not the bacterial) enzymes. Moreover, nucleosides lacking the N(3) undergo phosphorolysis and those lacking N(7) are inhibitors (but not substrates). Methylation of the ring N(7) leads to two overlapping effects: labilization of the glycosidic bond, and impediment to proton ation at this site by the enzyme, a postulated prerequisite for enzymatic phosphorolysis. It is proposed that a histidine interacts with N(1) as a don or and O6 as an acceptor. Alternatively N(1)−H and C(2)−NH2, may serve as donors for hydrogen bonds with a glutam ate residue. The less specific E. coli enzyme phosphorolyses all purine ring modified nucleosides but 7-deazainosine which is only an inhibitor. On the other hand, the bacterial enzyme exhibits decreased activity towards N(7)-methylated nucleosides and lack of affinity for a majority of the tested acyclonu cleoside inhibitors of the mammalian enzymes. The foregoing results underline the fundamental differences between mammalian and bacterial enzymes, including variations in the binding sites for the purine ring.

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