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 bifunction al dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypano soma cruzi. The DHFR domain was purified to homogeneity by methotrexat e-Sepharose chromatography followed by an anion-exchange chromatograph y step in a mono Q column, and displayed a single 27-kDa band on SDS-P AGE, Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the w ild-type bifunctional enzyme with K-m values of 0.75 mu M for dihydrof olate and 16 mu M for NADPH and a k(cat) value of 16.5 s(-1). T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the in hibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors th at form an initial complex which isomerizes slowly to a tighter comple x and are referred to as 'slow, tight-binding' inhibitors. While the s low-binding step of inhibition was apparently unaffected in the indivi dually 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.