scholarly journals Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase: Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase

1999 ◽  
Vol 344 (2) ◽  
pp. 585-592 ◽  
Author(s):  
Jason T. MAYNES ◽  
W.-S. YAM ◽  
Jack P. JENUTH ◽  
R. Gang YUAN ◽  
Steven A. LITSTER ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Hydrogen Bond ◽  
Purine Nucleoside Phosphorylase ◽  
Fold Increase ◽  
Purine Nucleoside ◽  
Dynamics Simulation ◽  
Amino Group ◽  
Wild Type ◽  
Nucleoside Phosphorylase ◽  
Purine Ring

Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in Km for inosine from 58 to 484 μM and a 1000-fold decrease in kcat/Km. The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a Km of 45 μM and a kcat/Km 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in kcat/Km, 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in Km with inosine and 7-fold decrease in kcat/Km as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a Km of 42 μM and a kcat/Km twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 Å, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 Å for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S0.5 for phosphate with wild type and the Asn-243-Asp enzymes were 1.35±0.01 and 1.84±0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.

Atomic Dissection of the Hydrogen Bond Network for Transition-State Analogue Binding to Purine Nucleoside Phosphorylase†

Biochemistry ◽  
2002 ◽  
Vol 41 (49) ◽  
pp. 14489-14498 ◽  
Author(s):  
Greg A. Kicska ◽  
Peter C. Tyler ◽  
Gary B. Evans ◽  
Richard H. Furneaux ◽  
Wuxian Shi ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Transition State ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Hydrogen Bond Network ◽  
Nucleoside Phosphorylase ◽  
Transition State Analogue ◽  
Bond Network

scholarly journals Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir

Biochimie ◽  
2012 ◽  
Vol 94 (1) ◽  
pp. 155-165 ◽  
Author(s):  
Rafael A. Caceres ◽  
Luís F.S.M. Timmers ◽  
Rodrigo G. Ducati ◽  
Diego O.N. da Silva ◽  
Luiz A. Basso ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Mycobacterium Tuberculosis ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase

scholarly journals Transport of adenine, hypoxanthine and uracil into Escherichia coli

1977 ◽  
Vol 168 (2) ◽  
pp. 195-204 ◽  
Author(s):  
K Burton
Keyword(s):  
Escherichia Coli ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Protonmotive Force ◽  
Wild Type ◽  
Nucleoside Phosphorylase ◽  
Uracil Phosphoribosyltransferase ◽  
Genetic Deficiency ◽  
Intracellular Metabolism ◽  
Type Strains

Uptake of adenine, hypoxanthine and uracil by an uncA strain of Escherichia coli is inhibited by uncouplers or when phosphate in the medium is replaced by less than 1 mM-arsenate, indicating a need for both a protonmotive force and phosphorylated metabolites. The rate of uptake of adenine or hypoxanthine was not markedly affected by a genetic deficiency of purine nucleoside phosphorylase. In two mutants with undetected adenine phosphoribosyltransferase, the rate of adenine uptake was about 30% of that in their parent strain, and evidence was obtained to confirm that adenine had then been utilized via purine nucleoside phosphorylase. In a strain deficient in both enzymes adenine uptake was about 1% of that shown by wild-type strains. Uptake of hypoxanthine was similarly limited in a strain lacking purine nucleoside phosphorylase, hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase. Deficiency of uracil phosphoribosyltransferase severely limits uracil uptake, but the defect can be circumvented by addition of inosine, which presumably provides ribose 1-phosphate for reversal of uridine phosphorylase. The results indicate that there are porter systems for adenine, hypoxanthine and uracil dependent on a protonmotive force and facilitated by intracellular metabolism of the free bases.

Molecular dynamics studies of a hexameric purine nucleoside phosphorylase

2009 ◽  
Vol 16 (3) ◽  
pp. 543-550 ◽  
Author(s):  
Fernando Berton Zanchi ◽  
Rafael Andrade Caceres ◽  
Rodrigo Guerino Stabeli ◽  
Walter Filgueira de Azevedo
Keyword(s):  
Molecular Dynamics ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase

scholarly journals Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine

2010 ◽  
Vol 169 (3) ◽  
pp. 379-388 ◽  
Author(s):  
Rafael Andrade Caceres ◽  
Luis Fernando Saraiva Macedo Timmers ◽  
Ivani Pauli ◽  
Lisandra Marques Gava ◽  
Rodrigo Gay Ducati ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase

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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