LEISHMANIA-MAJOR PTERIDINE-REDUCTASE-1 BELONGS TO THE SHORT-CHAIN DEHYDROGENASE FAMILY - STEREOCHEMICAL AND KINETIC EVIDENCE

Pteridine reductase 1 (PTR1) is a novel broad spectrum enzyme of pteri n and folate metabolism in the protozoan parasite Leishmania. Overexpr ession of PTR1 confers methotrexate resistance to these protozoa, aris ing from the enzyme's ability to reduce dihydrofolate and its relative insensitivity to methotrexate. The kinetic mechanism and stereochemic al course for the catalyzed reaction confirm PTR1's membership within the short chain dehydrogenase/reductase (SDR) family. With folate as a substrate, PTR1 catalyzes two rounds of reduction, yielding 5,6,7,8-t etrahydrofolate and oxidizing 2 equiv of NADPH. Dihydrofolate accumula tes transiently during folate reduction and is both a substrate and an inhibitor of PTR1, PTR 1 transfers the pro-S hydride of NADPH to carb on 6 on the si face of dihydrofolate, producing the same stereoisomer of THF as does dihydrofolate reductase. Product inhibition and isotope partitioning studies support an ordered ternary complex mechanism, wi th NADPH binding first and NADP(+) dissociating after the reduced pter idine. Identical kinetic mechanisms and NAD(P)H hydride chirality pref erences are seen with other SDRs. An observed tritium effect upon V/K for reduction of dihydrofolate arising from isotopic substitution of t he transferred hydride was suppressed at a high concentration of dihyd rofolate, consistent with a steady-state ordered kinetic mechanism. In terestingly, half of the binary enzyme-NADPH complex appears to be inc apable of rapid turnover. Fluorescence quenching results also indicate the existence of a nonproductive binary enzyme-dihydrofolate complex. The nonproductive complexes observed between PTR1 and its substrates are unique among members of the SDR family and may provide leads for d eveloping antileishmanial therapeutics.