Unexpected formation of an epoxide-derived multisubstrate adduct inhibitoron the active site of GAR transformylase

Multisubstrate adduct inhibitors (MAI) of glycinamide ribonucleotide transf ormylase (GAR Tfase), which incorporate key features of the folate cofactor and the beta -GAR substrate, typically exhibit K-i's in the picomolar rang e. However, these compounds have reduced bioavailability due to the incorpo ration of a negatively charged phosphate moiety that prevents effective cel lular uptake. Thus, a folate analogue that is capable of adduct formation w ith the substrate on the enzyme active site could lead to a potent GAR Tfas e inhibitor that takes advantage of the cellular folate transport systems. We synthesized a dibromide folate analogue, 10-bromo-10-bromomethyl-5,8,10- trideazafolic acid, that was an intermediate designed to assemble with the substrate beta -GAR on the enzyme active site. We have now determined the c rystal structure of the Escherichia coli GAR Tfase/MAI complex at 1.6 Angst rom resolution to ascertain the nature and mechanism of its time-dependent inhibition. The high-resolution crystal structure clearly revealed the exis tence of a covalent adduct between the substrate beta -GAR and the folate a nalogue (K-i = 20 muM). However, the electron density map surprisingly indi cated a C10 hydroxyl in the adduct rather than a bromide and suggested that the multisubstrate adduct is not formed directly from the dibromide but pr oceeds via an epoxide. Subsequently, we demonstrated the in situ conversion of the dibromide to the epoxide. Moreover, synthesis of the authentic epox ide confirmed that its inhibitory, time-dependent, and cytotoxic properties are comparable to those of the dibromide. Further, inhibition was stronges t when the dibromide or epoxide is preincubated with both enzyme and substr ate, indicating that inhibition occurs via the enzyme-dependent formation o f the multisubstrate adduct. Thus, the crystal structure revealed the succe ssful formation of an enzyme-assembled multisubstrate adduct and highlighte d a potential application for epoxides, and perhaps aziridines, in the desi gn of efficacious GAR Tfase inhibitors.