Abstract:
: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 (H4PteGlu5) so tightly that it does not dissociate during size exclusion chromatography. 10-Formyltetrahydrofolate dehydrogenase is also known to exhibit strong product inhibition by H4PteGlu5. There is a several-fold excess of 10-formyltetrahydrofolate dehydrogenase subunits in liver relative to the concentration of H4PteGlun, suggesting that in vivo this enzyme may bind significant amounts of this coenzyme in a nearly irreversible enzyme. H4PteGlu5 complex. How this tightly bound H4PteGlun is transferred to the other two enzymes in the cytosol, serine hydroxymethyltransferase and C1-tetrahydrofolate synthase, which use H4PteGlu5 as a substrate, is the subject of this investigation. Analysis of the product inhibition curve for 10-formyltetrahydrofolate dehydrogenase shows that H4-PteGlu5 has a dissociation constant near 15 nM which is 60-fold lower than the Ks for 10-formyl-H4PteGlu5. Fluorescence titration studies also yield a Kd of about 20 nM for H4PteGlu5. Coupling the 10-formyltetrahydrofolate dehydrogenase reaction to an excess of either serine hydroxymethyltransferase or C1-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 H4PteGlu5 bound per subunit. However, addition of either serine hydroxymethyltransferase or C1-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 H4PteGlu5.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Kim DW,Huang T,Schirch D,Schirch Vdoi
10.1021/bi9619684subject
Has Abstractpub_date
1996-12-10 00:00:00pages
15772-83issue
49eissn
0006-2960issn
1520-4995pii
bi9619684journal_volume
35pub_type
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