Reche, Pedro AArrebola, RSanti, D VGonzález-Pacanowska, D.Ruiz Pérez, Luis Miguel2023-06-202023-06-2019950166-6851https://hdl.handle.net/20.500.14352/58260We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.engjournal articlehttp://www.elsevier.com/wps/find/journaldescription.cws_home/506086/descriptionopen access612.017577.2Trypanosoma cruziDihydrofolate reductaseProtozoal enzymeHeterologous expressionFolate metabolismBioquímica (Biología)Microbiología (Biología)Biología molecular (Biología)2302 Bioquímica2414 Microbiología2415 Biología Molecular