WATER-MEDIATED SUBSTRATE PRODUCT DISCRIMINATION - THE PRODUCT COMPLEXOF THYMIDYLATE SYNTHASE AT 1.83-ANGSTROM/

In an irreversible enzyme-catalyzed reaction, strong binding of the pr oducts would lead to substantial product inhibition. The X-ray crystal structure of the product complex of thymidylate synthase (1.83-Angstr om resolution, R factor = 0.183 for all data between 7.0 and 1.83 Angs trom) identifies a bound water molecule that serves to disfavor bindin g of the product nucleotide, dTMP. This water molecule is hydrogen bon ded to absolutely conserved Tyr 146 (using the Lactobacillus casei num bering system) and is displaced by the C7 methyl group of the reaction product thymidylate. The relation between this observation and kineti c and thermodynamic values is discussed. The structure reveals a carba mate modified N-terminus that binds in a highly conserved site, replac ed by side chains that can exploit the same site in other TS sequences . The enzyme-products complex is compared to the previously determined structure of enzyme-substrate-cofactor analog. This comparison reveal s changes that occur between the first covalent complex formed between enzyme and substrate with an inhibitory cofactor analog and the compl eted reaction. The almost identical arrangement of ligands in these tw o structures contributes to our model for the TS reaction and verifies the physiological relevance of the mode in which potent inhibitors bi nd to this target for rational drug design.