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 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 cross-linked 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.
Fu
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TF
Institute of Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia, 23219, USA.
Maras
B
B
Barra
D
D
Schirch
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002845:Chromatography; D005492:Folic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000502:physiology; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013763:Tetrahydrofolates / Q000737:chemistry*; D013997:Time Factors; D000099295:Formyltetrahydrofolate Dehydrogenase
D005492:Folic Acid; D000587:Oxidoreductases Acting on CH-NH Group Donors; D013763:Tetrahydrofolates; D000099295:Formyltetrahydrofolate Dehydrogenase; C030371:5,6,7,8-tetrahydrofolic acid; C092738:10-formyl-5,8-dideazafolate