10-Formyltetrahydrofolate dehydrogenase has previously been identified as a tight binding protein of the polyglutamate forms of tetrahydrofolate (R. J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982). Each subunit contains two independently folded domains connected by a linking peptide. By using the stable substrate and product analogs 10-formyl 5,8-dideazafolate and 5,8-dideazafolate, respectively, we have determined that the tight binding folate site is separate from the catalytic site and that it is located on the N-terminal domain of the protein. This was achieved by cross- linking 10-formyl 5,8-dideazafolate to the dehydrogenase through the carboxyl group of the substrate analog. The cross-linked substrate analog was converted to the cross-linked product complex by adding either NADP+ or 2- mercaptoethanol, proving that the 10-formyl 5,8-dideazafolate was bound at the active site. With the active site crosslinked to 5,8-dideazafolate and not available for binding, the enzyme still bound 5,8-dideazafolate- [3H]tetraglutamate tightly but noncovalently. Separation of the large and small domains by limited proteolysis showed that the tightly bound 5,8- dideazafolate-[3H]tetraglutamate was located on the small domain. The location of the cross-linked 10-formyl 5,8-dideazafolate at the active site was determined by amino acid sequencing of an isolated tryptic peptide.
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