Open AccessInhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy
Author(s) -
VERES Zsuzsa,
NESZMÉLYI András,
SZABOLCS Anna,
DÉNES Géza
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb14441.x
Subject(s) - phosphorolysis , thymidine phosphorylase , pyrimidine , glycosidic bond , chemistry , purine nucleoside phosphorylase , stereochemistry , uridine , thymidine , uracil , glycogen phosphorylase , nucleoside , substrate (aquarium) , enzyme , biochemistry , rna , dna , purine , biology , ecology , gene
Some 3′‐ and/or 5′‐substituted pyrimidine nucleosides, as well as anhydropyrimidine nucleosides, which have no flexibility about the N ‐glycosidic bond were studied as inhibitors of thymidine phosphorylase and uridine phosphorylase. The conformation of some analogs was also investigated in order to obtain information on substrate binding to the enzyme. The above compounds, including the potential anti‐(human immunodeficiency virus) agent, 3′‐azido‐2′,3′‐dideoxy‐5‐methyluridine were not substrates for either thymidine phosphorylase or uridine phosphorylase. (The only exception was arabinofuranosyl‐5‐ethyluracil, which proved to be a poor substrate for uridine phosphorylase). The phosphorolysis of thymidine by thymidine phosphorylase was slightly or not at all altered by these pyrimidine nucloside analogs. The lowest K i was obtained in the case of 3′‐azido‐2′,3′‐dideoxy‐5‐methyluridine and the highest in the case of 2′‐deoxylyxofuranosyl‐5‐ethyluracil, when studying the analogs with flexible structure as inhibitors of uridine phosphorylase. The K i for 2,3′‐ and 2,5′‐anhydro‐2′‐deoxy‐5‐ethyluridine was 5–6 orders of magnitude higher than that for 2,2′‐anhydro‐5‐ethyluridine. Competitive inhibition was observed in all cases. For these three molecules computer‐aided molecular modelling predicts the following glycosidic torsion angles X (O 4 ,‐C 1 ,‐N 1 ‐C 2 ): 109° for 2,2′‐anhydro‐5‐ethyluridine, and 78° and 71° for 2,3′‐ and 2,5′‐anhydro‐2′‐deoxy‐5‐ethyluridine respectively. These values are corroborated by high‐resolution 13 C‐ and 1 H‐NMR studies. 2′‐Deoxy‐5‐ethyluridine is predicted to have a syn conformation with X = 46° and Δ E about 2.5 kJ/mol over the minimum energy (in anti position, X =−147°). 1 H and 13 C data including homonuclear Overhauser enhancements complete the information about the solution conformation. Considering the K i values obtained, it is likely that substrates of uridine phosphorylase will bind to the enzyme in the same conformation as 2,2′‐anhydro‐5‐ethyluridine. The >30° deviation from the N ‐glycosidic torsion angle of 2,2′‐anhydro‐5‐ethyluridine results in much higher K i values.