AN ESSENTIAL ROLE FOR WATER IN AN ENZYME REACTION-MECHANISM - THE CRYSTAL-STRUCTURE OF THE THYMIDYLATE SYNTHASE MUTANT E58Q

A water-mediated hydrogen bond network coordinated by glutamate 60(58) appears to play an important role in the thymidylate synthase (TS) re action mechanism. We have addressed the role of glutamate 60(58) in th e TS reaction by cocrystallizing the Escherichia coli TS mutant E60(58 )Q with dUMP and the cofactor analog CB3717 and have determined the X- ray crystal structure to 2.5 Angstrom resolution with a final R factor of 15.2% (R(free) = 24.0%). Using difference Fourier analysis, we ana lyzed directly the changes that occur between the wild-type and mutant structures, The structure of the mutant enzyme suggests that E60(58) is not required to properly position the ligands in the active site an d that the coordinated hydrogen bond network has been disrupted in the mutant, providing an atomic resolution explanation for the impairment of the TS reaction by the E60(58)Q mutant and confirming the proposal that E60(58) coordinates this conserved hydrogen bond network. The st ructure also provides insight into the role of specific waters in the active site which have been suggested to be important in the TS reacti on. Finally, the structure shows a unique conformation for the cofacto r analog, CB3717, which has implications for structure-based drug desi gn and sheds light on the controversy surrounding the previously obser ved enzymatic nonidentity between the chemically identical monomers of the TS dimer.