 It has been proven that the imine formed through reaction with THF and dUMP is an intermediate in the reaction with dUMP through mutations in the structure of TS that inhibit the completion of the mechanism.V316Am TS, a mutant with deletion of C terminal valines from both subunits, allows the catalysis of dehalogenation of BrdUMP preceding the mechanism described above and the covalent bond to THF and dUMP.The mutant TS is unable to accomplish the C-terminal conformational change needed to break covalent bonds to form dTMP, thus showing the proposed mechanism to be true.