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Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes
Author(s) -
Yoneyama Toshie,
Hatakeyama Kazuyuki
Publication year - 2001
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.38501
Subject(s) - gtp cyclohydrolase i , gtp' , phenylalanine , tetrahydrobiopterin , biochemistry , chemistry , phenylalanine hydroxylase , ligand (biochemistry) , binding site , biophysics , stereochemistry , cofactor , enzyme , biology , receptor , amino acid
GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6 R ‐ l ‐ erythro ‐5,6,7,8‐tetrahydrobiopterin (BH 4 ), which is an essential cofactor for key enzymes producing catecholamines, serotonin, and nitric oxide as well as phenylalanine hydroxylase. GFRP also mediates feed‐forward stimulation of GTP cyclohydrolase I activity by phenylalanine at subsaturating GTP levels. These ligands, BH 4 and phenylalanine, induce complex formation between one molecule of GTP cyclohydrolase I and two molecules of GFRP. Here, we report the analysis of ligand binding using the gel filtration method of Hummel and Dreyer. BH 4 binds to the GTP cyclohydrolase I/GFRP complex with a K d of 4 μM, and phenylalanine binds to the protein complex with a K d of 94 μM. The binding of BH 4 is enhanced by dGTP. The binding stoichiometrics of BH 4 and phenylalanine were estimated to be 10 molecules of each per protein complex, in other words, one molecule per subunit of protein, because GTP cyclohydrolase I is a decamer and GFRP is a pentamer. These findings were corroborated by data from equilibrium dialysis experiments. Regarding ligand binding to free proteins, BH 4 binds weakly to GTP cyclohydrolase I but not to GFRP, and phenylalanine binds weakly to GFRP but not to GTP cyclohydrolase I. These results suggest that the overall structure of the protein complex contributes to binding of BH 4 and phenylalanine but also that each binding site of BH 4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.