ISOLATION OF A COVALENT STEADY-STATE INTERMEDIATE IN GLUTAMATE-60 MUTANTS OF THYMIDYLATE SYNTHASE

Glutamate 60 of thymidylate synthase coordinates a hydrogen bond netwo rk important in proton transfer reactions to and from the substrate dU MP. The E60A and E60L mutants of Lactobacillus casei thymidylate synth ase catalyzed tritium exchange from [5-H-3]dUMP for solvent protons fa ster than dTMP formation, indicating accumulation of a steady-state in termediate and a change in partitioning of the intermediate. A covalen t complex consisting of E60A or E60L thymidylate synthase, dUMP, and t he cofactor CH(2)H(4)folate was isolated on SDS-polyacrylamide gel ele ctrophoresis and shown to be chemically and kinetically competent to f orm dTMP. These results provide proof of the formation of a covalent s teady-state intermediate in the reaction pathway of thymidylate syntha se and demonstrate that the rate-determining step in the mutants occur s during conversion of the covalent intermediate to dTMP.