Inactivity of N229A thymidylate synthase due to water-mediated effects: Isolating a late stage in methyl transfer

Mutation of thymidylate synthase N229(177) to alanine results in an essenti ally inactive enzyme, yet it leads to formation of a stable ternary complex . The kinetics of N229(177)A show that k(cat) for Escherichia coli is reduc ed by 200-fold while the K-m for dUMP is increased 200-fold and the K-m for folate increased by tenfold versus the wild-type enzyme. The crystal struc tures of N229(177)A in complex with dUMP and CB3717, and in complex with dU MP alone are determined at 2.4 Angstrom, and 2.5 Angstrom resolution. These structures identify the covalently bound ternary complex and show how N229 (177)A traps an intermediate, and so becomes inactive in a later step of th e reaction. Since the smaller alanine side-chain at N229(177)A does not dir ectly sterically impair binding of ligands, the structures implicate, and p lace quantitative limits on the involvement of the structured water network in the active site of thymidylate synthase in both catalysis and in determ ining the binding affinity for dUMP (in contrast, the N229(177)V mutation i n Lactobacillus casi has minimal effect on activity). (C) 1998 Academic Pre ss.