Tetrahydrofolate-dependent Labilization of the Hydrogen Atom on Carbon 5 of 5'-Deoxycytidylate, a Step in the Deoxycytidylate Hydroxymethylase Reaction

Abstract

Abstract Deoxycytidylate hydroxymethylase, induced by the infection of Escherichia coli by T4 bacteriophage, specifically catalyzes an exchange between the hydrogen atom on carbon 5 of dCMP and water. The exchange is conveniently measured by the release of tritium from dCMP-5-3H into water. While tetrahydrofolate is required for the exchange, formaldehyde has been shown not to be involved. This exchange was not detectable in crude extracts of E. coli, but was found only in extracts of infected cells. The exchange activity was absent in extracts of cells infected by amber mutants of T4 which do not show dCMP hydroxymethylase activity. During purification of dCMP hydroxymethylase, the ratio of the rate of the over-all reaction to the rate of displacement of tritium from dCMP-5-3H remained constant. The rates of displacement of tritium from dCMP-5-3H, the incorporation of tritium ions from water into dCMP, and the incorporation of formaldehyde into hydroxymethyl dCMP have been compared. From these and other data, it has been concluded that the dCMP hydroxymethylase reaction consists of two reversible steps: the first, a tetrahydrofolate-dependent displacement of the hydrogen on carbon 5 of dCMP, and the second, the 5-hydroxymethylation of dCMP by formaldehyde or a derivative of formaldehyde, perhaps 5,10-methylene tetrahydrofolate. The first step has been postulated to be a reaction between carbon 5 of dCMP and the enzyme. The relationship of the hydroxymethylase system to the thymidylate synthetase reaction has been discussed. The release of tritium from dCMP-5-3H is a specific and sensitive assay for dCMP hydroxymethylase. dCMP hydroxymethylase induced by the infection of E. coli with bacteriophage T4 exhibited essentially the same properties during purification as the enzyme induced by phage T6.