Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase

Biochemistry ◽  
2016 ◽  
Vol 55 (14) ◽  
pp. 2153-2162 ◽  
Author(s):  
Geir Villy Isaksen ◽  
Kathrin Helen Hopmann ◽  
Johan Åqvist ◽  
Bjørn Olav Brandsdal
Keyword(s):  
Substrate Specificity ◽  
Computer Simulations ◽  
Purine Nucleoside Phosphorylase ◽  
Bond Cleavage ◽  
Purine Nucleoside ◽  
Glycosidic Bond ◽  
Nucleoside Phosphorylase ◽  
Glycosidic Bond Cleavage

COUPLING PROTEIN DYNAMICS TO REACTION CENTER ELECTRON DENSITY IN ENZYMES: AN ELECTRONIC PROTEIN PROMOTING VIBRATION IN HUMAN PURINE NUCLEOSIDE PHOSPHORYLASE

2004 ◽  
Vol 03 (04) ◽  
pp. 501-509 ◽  
Author(s):  
JOSHUA S. MINCER ◽  
SARA NUÑEZ ◽  
STEVEN D. SCHWARTZ
Keyword(s):  
Protein Dynamics ◽  
Barrier Height ◽  
Active Site ◽  
Purine Nucleoside Phosphorylase ◽  
Bond Cleavage ◽  
Purine Nucleoside ◽  
Active Site Residue ◽  
Charge Fluctuations ◽  
Nucleoside Phosphorylase ◽  
Coupling Protein

The notable three oxygen stacking that occurs upon binding of ribonucleoside substrate and phosphate nucleophile by human purine nucleoside phosphorylase (hPNP) enables the coupling of protein dynamic modes to compress this stack, squeezing the ribosyl O4' between ribosyl O5' and the nuclophilic O P . Created primarily by the motion of active site residue H257, this compression dynamically lowers the barrier height for N9–C1' ribosidic bond cleavage by as much as 20%. As such, this compression constitutes a protein promoting vibration (PPV) (S. Nuñez et al.). Presently, we demonstrate charge fluctuations in the ribose and purine components of the ribonucleoside substrate, as well as specifically across the N9–C1' ribosidic bond, that are correlated with the PPV and can explain the decrease in reaction barrier height due to their facilitating cleavage of the ribosidic bond. hPNP apparently employs protein dynamics to push electrons, a finding that suggests that this coupling may be found more generally in enzymes that catalyze substitution and elimination reactions.

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).

Purine Nucleoside Phosphorylase (PNP) Deficiency Leading to Accumulation of Lymphocytes in S-Phase

1986 ◽  
Vol 3 (4) ◽  
pp. 353-359 ◽  
Author(s):  
Ger T. Rijkers ◽  
Ben J. M. Zegers ◽  
Leo J. M. Spaapen ◽  
Derk H. Rutgers ◽  
John J. Roord ◽  
...  
Keyword(s):  
Purine Nucleoside Phosphorylase ◽  
S Phase ◽  
Purine Nucleoside ◽  
Nucleoside Phosphorylase
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