On the mechanism of the acid-catalyzed hydrolysis of uridine to uracil. Evidence for 6-hydroxy-5,6-dihydrouridine intermediates.

Abstract

In acidic media, the 5,6-double bond of uridine is rapidly hydrated to give a small amount of 6-hydroxy-5,6-dihydrouridine (Urd-H2O), the mechanism of which is known from studies of the acid-catalyzed dehydration of Urd-H2O (Prior, J. J., Maley, J., and Santi, D. V. (1984) J. Biol. Chem. 258, 2422-2428). In addition to dehydration, Urd-H2O also undergoes direct hydrolysis of the N-glycosidic bond in acidic solution. The kinetics of the above reaction demonstrates that Urd-H2O, or an intermediate in the pathway leading from Urd to Urd-H2O, is kinetically competent to account for the hydrolysis of the N-glycosidic bond of Urd. The hydrolysis of (1'-2H)Urd proceeds with an alpha-secondary deuterium isotope effect of kH/kD of 1.11 at 25 degrees C. This isotope effect is sufficiently large to implicate carbonium ion character at the 1'-carbon during hydrolysis but, since it is not the maximal value expected, suggests that N-glycoside cleavage is rate-determining with a transition state intermediate between reactant and products. Importantly, the hydrolysis of [6-3H]Urd proceeds with a substantial inverse secondary isotope effect of kT/kH = 1.15 at 25 degrees C which indicates some degree of sp2 to sp3 rehybridization of C-6 of the pyrimidine moiety during hydrolysis. From the data available, it appears that an important pathway in the hydrolysis of the N-glycoside bond of Urd involves either spontaneous cleavage of Urd which is protonated at the 5-carbon or a protonated species of Urd-H2O. The studies described here, together with the known susceptibility of the 6-position of pyrimidine heterocycles toward nucleophiles, permits the proposal of chemically reasonable mechanisms for enzyme-catalyzed cleavage of N-glycosidic bonds of pyrimidines.

• Authors: J J Prior, D V Santi
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 259 Issue 4 Pg. 2429-34 (Feb 25 1984) ISSN: 0021-9258 [Print] United States
• PMID: 6546573 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Carbon Radioisotopes
• Glycosides
• Tritium
• 6-hydroxy-5,6-dihydrouridine
• Uracil
• Uridine

Topics

• Carbon Radioisotopes
• Chemical Phenomena
• Chemistry
• Glycosides
• Hydrogen-Ion Concentration
• Hydrolysis
• Kinetics
• Tritium
• Uracil
• Uridine (analogs & derivatives)