Cysteine-catalyzed Hydrogen Isotope Exchange at Position 5 of Uridylic Acid

Abstract

A new and potentially useful method for labeling nucleic acid was developed.Cysteine catalyzes hydrogen-deuterium exchange at position 5 of uridine 5'-phosphate under mild conditions. The pD dependence of the catalysis by cysteine, as well as that by 2-mercaptoethylamine showed a maximum at pD 8.9. Typical rates of the exchange at 37°C, determined by NMR spectroscopy were as follows: Reagent and its concentration/pD/the apparent rate constant (hr^-1); cysteine, 0.5 m/9.0/2.29×10^-2;cysteine, 1.0 M/9.0/6.05×10^-2; 2-mercaptoethylamine, 0.5 M/9.1/1.26×10^-2; 2-mercaptoethanol, 0.5 M/9.5/1.51×10^-3. N-Acetylcysteine, S-methylcysteine, or a mixture of the two agents was essentially ineffective in catalysis of the exchange. The amino group of cysteine is important in the catalysis since cysteine was much more effective than 2-mercaptoethanol as catalyst with 3-methyluridine at pD 10.0 where the SH-group of both compounds is largely dissociated. Based on these data, together with the known roles of a sulfur nucleophile and of amine in the bisulfite-catalyzed hydrogen isotope exchange of uridine (Y. Wataya and H. Hayatsu, Biochemistry, 11, 3583 (1972)), a reaction mechanism is proposed in which the mercapto anion adds across the 5, 6-double bond of the undissociated uracil moiety forming a 5, 6-dihydrouridine-6-mercapto compound and the NH₂-group of cysteine abstracts the hydrogenat the 5-position intramolecularly. Studies on the effect of added amines and on the dependence of the exchange rate on the cysteine concentration suggested that protonated amines, either of added amine or of cysteine itself, accelerate the overall reaction. The acceleration is probably due to promotion of the addition of the SH to uridine. The implications of these findings in relation to the mechanism of the thymidylate synthetase reaction are discussed. This catalysis by cysteine appears to be the mildest method so far known for labeling uracil.