New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with beta-GAR and 10-formyl-5,8,10-trideazafolic acid

The crystal structure of Escherichia coli GAR Tfase at 2.1 Angstrom resolut ion in complex with 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, K- i = 260 nM), an inhibitor designed to form an enzyme-assembled multisubstra te adduct with the substrate, beta-GAR, was studied to determine the exact nature of its inhibitory properties. Rather than forming the expected coval ent adduct, the folate inhibitor binds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedral intermed iate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpected insights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between 10-formyl-TDA F and the corresponding alcohol, and a further 10-fold difference for inhib itors that lack the alcohol. The presence of the hydrated aldehyde was conf irmed in solution by C-13-H-1 NMR spectroscopy of the ternary GAR Tfase-bet a-GAR-10-formyl-TDAF complex using the C-13-labeled-10-formyl-TDAF. This in sight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors, provides a novel and previously unrecognized basi s for the design of more potent inhibitors of the folate-dependent formyl t ransfer enzymes of the purine biosynthetic pathway and development of anti- neoplastic agents.