Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain.
We 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.
Reche
P
P
Instituto de Parasitología y Biomedicina, Granada, Spain.
Arrebola
R
R
Santi
D V
DV
Gonzalez-Pacanowska
D
D
Ruiz-Perez
L M
LM
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D017125:Genes, Protozoan; D008727:Methotrexate / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D011739:Pyrimethamine / Q000037:antagonists & inhibitors; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014295:Trimethoprim / Q000037:antagonists & inhibitors; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate; D011739:Pyrimethamine