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

Tzu-Fun Fu, Bruno Maras, Donatella Barra, Verne Schirch

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)161-166
Number of pages6
JournalArchives of Biochemistry and Biophysics
Volume367
Issue number2
DOIs
Publication statusPublished - 1999 Jul 15

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Catalytic Domain
Binding Sites
Substrates
Proteolysis
Polyglutamic Acid
Peptides
Biochemistry
Mercaptoethanol
Protein Sequence Analysis
Binding sites
NADP
Folic Acid
Crosslinking
Carrier Proteins
Oxidoreductases
Amino Acids
10-formyl-5,8-dideazafolate
formyltetrahydrofolate dehydrogenase
5,6,7,8-tetrahydrofolic acid
Enzymes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

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title = "A noncatalytic tetrahydrofolate tight binding site is on the small domain of 10-formyltetrahydrofolate dehydrogenase",
abstract = "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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A noncatalytic tetrahydrofolate tight binding site is on the small domain of 10-formyltetrahydrofolate dehydrogenase. / Fu, Tzu-Fun; Maras, Bruno; Barra, Donatella; Schirch, Verne.

In: Archives of Biochemistry and Biophysics, Vol. 367, No. 2, 15.07.1999, p. 161-166.

Research output: Contribution to journalArticle

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T1 - A noncatalytic tetrahydrofolate tight binding site is on the small domain of 10-formyltetrahydrofolate dehydrogenase

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AB - 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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