A noncatalytic tetrahydrofolate tight binding site is on the small domain of 10-formyltetrahydrofolate dehydrogenase

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 conta ins two independently folded domains connected by a linking peptide. By usi ng 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 o f the substrate analog. The cross-linked substrate analog was converted to the cross-linked product complex by adding either NADP(+) or 2-mercaptoetha nol, proving that the 10-formyl 5,8-dideazafolate was bound at the active s ite. With the active site crosslinked to 5,8-dideazafolate and not availabl e for binding, the enzyme still bound 5,8-dideazafolate-[H-3]tetraglutamate tightly but noncovalently. Separation of the large and small domains by li mited proteolysis showed that the tightly bound 5,8-dideazafolate-[H-3]tetr aglutamate was located on the small domain. The location of the cross-linke d 10-formyl 5,8-dideazafolate at the active site was determined by amino ac id sequencing of an isolated tryptic peptide, (C) 1999 Academic Press.