Glutathione-Catalyzed Hydrogen Isotope Exchange at Position 5 of Uridine. a Model for Enzymic Carbon Alkylation Reactions of Pyrimidines

The effect of glutathione (GSH) on the exchange of hydrogen to deuterium at position 5 of uridine (Urd) was studied by using proton magnetic resonance spectroscopy. It was found that in D20 solutions, at 80°, the rate of H-isotope exchange was enhanced in the presence of GSH and that the enhancement of the pseudo-first-order rate of exchange was proportional to the GSH concentration. The results obtained with GSH derivatives indicated the requirement of a free SH group for catalysis. The GSH-catalyzed H-isotope exchange showed a bell-shaped dependence on the OD^- ion concentration, suggesting that in the rate-determining step the ionized SH group of GSH reacts with the nonionized species of Urd. Ionization of Urd causes a substantial shielding of the proton at position 6, indicating the increased electron density of the 5,6-double bond, which may account for the lack of reactivity observed at high pD values. The results are consistent with a catalytic mechanism of H-isotope exchange involving the reversible addition elimination of the SH group of GSH across the 5,6-double bond of Urd. The relevance of these findings to the mechanism of enzyme-catalyzed C-alkylation reactions of pyrimidine nucleotides is discussed.