Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP.
The activity of GTP cyclohydrolase I is inhibited by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and stimulated by phenylalanine through complex formation with GTP cyclohydrolase I feedback regulatory protein (GFRP). Gel filtration experiments as well as enzyme activity measurements showed that the number of subunits of GFRP in both the inhibitory and stimulatory complexes is equal to that of GTP cyclohydrolase I. Because GFRP is a pentamer and GTP cyclohydrolase I was shown here by cross-linking experiments to be a decamer, the results indicate that two molecules of a pentameric GFRP associate with one molecule of GTP cyclohydrolase I. Gel filtration analysis suggested that the complex has a radius of gyration similar to that of the enzyme itself. These observations support our model that one molecule of GFRP binds to each of the two outer faces of the torus-shaped GTP cyclohydrolase I. For formation of the inhibitory protein complex, both BH4 and GTP were required; the median effective concentrations of BH4 and GTP were 2 and 26 microM, respectively. BH4 was the most potent of biopterins with different oxidative states. Among GTP analogues, dGTP as well as guanosine 5'-O-(3'-thiotriphosphate) exhibited similar inducibility compared with GTP, whereas other nucleotide triphosphates had no effect. On the other hand, phenylalanine alone was enough for formation of the stimulatory protein complex, and positive cooperativity was found for the phenylalanine-induced protein complex formation. Phenylalanine was the most potent of the aromatic amino acids.
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
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D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002850:Chromatography, Gel / Q000379:methods; D003432:Cross-Linking Reagents / Q000378:metabolism; D004120:Dimethyl Suberimidate / Q000378:metabolism; D006136:GTP Cyclohydrolase / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D010649:Phenylalanine / Q000494:pharmacology*; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry
D003432:Cross-Linking Reagents; C491065:Gchfr protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011994:Recombinant Proteins; D001708:Biopterins; D004120:Dimethyl Suberimidate; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D006136:GTP Cyclohydrolase; C003402:sapropterin