Promoting Vibrations in Human Purine Nucleoside Phosphorylase. A Molecular Dynamics and Hybrid Quantum Mechanical/Molecular Mechanical Study

2004 ◽  
Vol 126 (48) ◽  
pp. 15720-15729 ◽  
Author(s):  
Sara Núñez ◽  
Dimitri Antoniou ◽  
Vern L. Schramm ◽  
Steven D. Schwartz
Keyword(s):  
Molecular Dynamics ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Quantum Mechanical ◽  
Nucleoside Phosphorylase ◽  
Mechanical Study

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 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.

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

Purine nucleoside phosphorylase: Isolation and characterization

1990 ◽  
Vol 55 (12) ◽  
pp. 2987-2999 ◽  
Author(s):  
Katarina Šedivá ◽  
Ivan Votruba ◽  
Antonín Holý ◽  
Ivan Rosenberg
Keyword(s):  
Molecular Weight ◽  
Purine Nucleoside Phosphorylase ◽  
Purine Nucleoside ◽  
Leukemia Cells ◽  
Ph Optimum ◽  
Nucleoside Phosphorylase ◽  
Isolation And Characterization ◽  
Reaction Proceeds ◽  
The Matrix ◽  
Sepharose 4B

Purine nucleoside phosphorylase (PNP) from mouse leukemia cells L1210 was purified to homogeneity by a combination of ion exchange and affinity chromatography using AE-Sepharose 4B and 9-(p-succinylaminobenzyl)hypoxanthine as the matrix and the ligand, respectively. The native enzyme has a molecular weight of 104 000 and consists of three subunits of equal molecular weight of 34 000. The results of isoelectric focusing showed that the enzyme is considerably microheterogeneous over the pI-range 4.0-5.8 and most likely consists of eight isozymes. The temperature and pH-optimum of phosphorolysis, purine nucleoside synthesis and also of transribosylation is identical, namely 55 °C and pH 7.4. The transribosylation reaction proceeds in the presence of phosphate only. The following Km-values (μmol l-1) were determined for phosphorolysis: inosine 40, 2'-deoxyinosine 47, guanosine 27, 2'-deoxyguanosine 32. The Km-values (μmol l-1) of purine riboside and deoxyriboside synthesis are lower than the values for phosphorolysis (hypoxanthine 18 and 34, resp., guanine 8 and 11, resp.). An affinity lower by one order shows PNP for (-D-ribose-1-phosphate, (-D-2-deoxyribose-1-phosphate (Km = 200 μmol l-1 in both cases) and phosphate (Km = 805 μmol l-1). The substrate specificity of the enzyme was also studied: positions N(1), C(2) and C(8) are decisive for the binding of the substrate (purine nucleoside).

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