TIGHT-BINDING OF FOLATE SUBSTRATES AND INHIBITORS TO RECOMBINANT MOUSE GLYCINAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE

The binding of the prototypical folate inhibitor of de novo purine syn thesis, 5,10-dideazatetrahydrofolate (DDATHF), and its hexaglutamate t o recombinant trifunctional mouse glycinamide ribonucleotide formyltra nsferase (rmGARFT) was studied by equilibrium dialysis and by steady-s tate kinetics using sensitive assays that allowed initial rate calcula tions, rmGARFT was expressed in insect cells infected with a recombina nt baculovirus and purified by a two-step procedure that allowed produ ction of about 25 mg of pure protein/L of culture. The binding of DDAT HF to GARFT was approximate to 50-fold tighter than previously reporte d, with K-d and K-i values of 2-9 nM, making the parent form of this a ntifolate a tight-binding inhibitor, The binding of the hexaglutamate of DDATHF to rmGARFT had K-d and K-i values of 0.1-0.3 nM, consistent with the view that polyglutamation enhances binding of antifolates to GARFT. Kinetic analyses using either mono-or hexaglutamate substrate d id not yield different values for the K-i for the hexaglutamate form o f DDATHF, in contradiction with previous reports, Both the folate subs trate commonly used to study GARFT, 10-formyl-5,8-dideazafolate, and i ts hexaglutamate were found to have very low K-m values, namely, 75 an d 7.4 nM, respectively, and the folate reaction products for these sub strates were equally potent inhibitors, results which modify the inter pretation of previous kinetic experiments. The product analog DDATHF a nd beta-glycinamide ribonucleotide bound to enzyme equally well in the presence and absence of the other, an observation at variance with th e concept that GARFT obeys an ordered sequential binding of the substr ates. We conclude that the kinetics of mouse GARFT are most consistent with a random order of substrate binding, that both the inhibitor DDA THF and the folate substrate are tight-binding ligands, and that polyg lutamate forms enhance the affinity of both substrate and inhibitor by an order of magnitude.