Properties of tetrahydropteroylpentaglutamate bound to 10-formyltetrahydrofolate dehydrogenase

A new rapid procedure for purifying 10-formyltetrahydrofolate dehydrogenase results in 90 mg of pure enzyme from two rabbit livers. This abundant liver enzyme is known to bind its product tetrahydropteroylpentaglutamate (H(4)PteGlu(5)) so tightly that it does not dissociate during size exclusion chromatography. 10-Formyltetrahydrofolate dehydrogenase is also known to exhibit strong product inhibition by H(4)PteGlu(5). There is a several-fold excess of 10-formyltetrahydrofolate dehydrogenase subunits in liver relative to the concentration of H(4)PteGlu(n), suggesting that in vivo this enzyme may bind significant amounts of this coenzyme in a nearly irreversible enzyme . H(4)PteGlu(5) complex. How this tightly bound H(4)PteGlu(n), is transferred to the other two enzymes in the cytosol, serine hydroxymethyltransferase and C-1-tetrahydrofolate synthase, which use H(4)PteGlu(5) as a substrate, is the subject of this investigation. Analysis of the product inhibition curve for 10-formyltetrahydrofolate dehydrogenase shows that H(4)PteGlu(5) has a dissociation constant near 15 nM which is 60-fold lower than the K-s for 10-formyl-H-4-PteGlu(5). Fluorescence titration studies also yield a K-d of about 20 nM for H(4)PteGlu(5). Coupling the 10-formyltetrahydrofolate dehydrogenase reaction to an excess of either serine hydroxymethyltransferase or C-1-tetrahydrofolate synthase not only abolishes product inhibition but also increases the initial rate of its activity by about 2-fold. Passage of a reaction mixture of 10-formyltetrahydrofolate dehydrogenase down a size exclusion column results in enzyme with 1 equiv of H(4)PteGlu(5) bound per subunit, However, addition of either serine hydroxymethyltransferase or C-1-tetrahydrofolate synthase results in a rapid transfer of this bound folate to these enzymes. Evidence is also presented that the tightly bound folate is in equilibrium with solvent H(4)PteGlu(5).