Model Studies of Thymidylate Synthetase. Intramolecular Catalysis of 5-Hydrogen Exchange and 5-Hydroxymethylation of 1-Substituted Uracils

Daniel V. Santi, Curtis F. Brewer

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Abstract

The mechanisms of base-catalyzed 5-hydrogen exchange and 5-hydroxymethylation of 1-substituted uracils have been examined to provide insight into the mechanism of the analogous reactions catalyzed by thymidylate synthetase. Kinetic studies of 5-H exchange demonstrate that the reaction requires attack of a nucleophile at the 6 position of the heterocycle to form carbanionic intermediates, as in classical addition reactions of α,β-unsaturated carbonyl compounds. The intermediate then accepts a proton from water to form the corresponding carbon acid; reversal of these steps results in 5-H exchange. Proton transfers at the 5 position of the saturated pyrimidine intermediates have been found to be susceptible to general acid-base catalysis and may be rate determining. The exchange reaction and 5-hydroxymethylation are greatly facilitated by intramolecular catalysis involving addition of a nucleophile attached to the 1 substituent to the 6 position of the heterocycle; in fact, the rate of intramolecular catalyzed exchange of one of the reactive ionic species of 2′,3′-O-isopropylideneuridine is comparable to that of the enzyme-catalyzed reaction. Depending upon the efficacy of the intramolecular catalyst the rate-determining step of the exchange may be either nucleophilic attack at the 6 position of the heterocycle or proton transfer reactions at the 5 position. From these results, a minimal mechanism for the thymidylate synthetase catalyzed 5-H exchange of 2′-deoxyuridylate and its condensation with 5,10-methylenetetrahydrofolic acid is proposed.

Original languageEnglish (US)
Pages (from-to)2416-2424
Number of pages9
JournalBiochemistry
Volume12
Issue number13
DOIs
Publication statusPublished - Jun 1 1973

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ASJC Scopus subject areas

  • Biochemistry

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