Thymidylate synthetase from Escherichia coli has been shown to catalyze an exchange between water and the hydrogen on carbon atom 5 of deoxyuridylate at a rate which is approximately 5 to 10% of the rate of the over-all reaction. The exchange reaction exhibited an absolute requirement only for tetrahydrofolate. In the absence of formaldehyde or of Mg++, the exchange activity was reduced to about 25 and 40%, respectively, of the activity of the complete system, whereas the rates of both the over-all reaction and the displacement of tritium from deoxyuridylate-5-3H were reduced to about 5 and 25%, respectively. These findings have been interpreted as support for the reversible formation of an intermediate having a methylene bridge between carbon atom 5 of deoxyuridylate and nitrogen atom 5 of tetrahydrofolate.
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Lomax et al. (1967) studied this question.