Light activates reduction of methotrexate by NADPH in the ternary complex with Escherichia coli dihydrofolate reductase

Methotrexate (MTX), a strong inhibitor of dihydrofolate reductase (DHFR), h as been widely used for chemotherapy for many types of cancer as well as fo r juvenile rheumatoid arthritis, It mimics folate substrates and binds tigh tly to the active site of DHFR, perhaps in a conformation close to the tran sition state of the folate catalyzed reaction. Absorption, fluorescence and ultrasensitive Raman difference spectroscopies show that light-activated M TX reacts with NADPH in the enzyme active site, producing 5,8-dihydromethot rexate (5,8-dihydro-MTX) and NADP(+). The reaction, which proceeds with a h ydride transfer between C4 (pro-R side) of the nicotinamide ring and N5 of the pteridine ring, is similar to that between folate and NADPH except that the hydride is transferred to C6 in this case. Hence, MTX is catalytically competent in its excited state. Most experiments were performed on the Esc herichia coli enzyme, but preliminary studies show that the reaction also o ccurs with human DHFR.