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.