GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits. Purification, cDNA cloning, and bacterial expression.
GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzyme activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromatography and isolated cDNA clones, based on partial amino acid sequences of peptides derived from purified GFRP. Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid sequence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated Mr of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with six consecutive histidine residues at its N terminus. The fusion protein was affinity-purified and digested with thrombin to remove the histidine tag. The resulting recombinant GFRP showed kinetic properties similar to those of GFRP purified from rat liver. Cross-linking experiments using dimethyl suberimidate indicated that GFRP was a pentamer of 52 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average Mr of 49,734, which is 5 times the calculated molecular weight of the recombinant GFRP polypeptide. Based on the pentameric structure of GFRP, we have proposed a model for the quaternary structure of GFRP and GTP cyclohydrolase I complexes.
Yoneyama
T
T
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Brewer
J M
JM
Hatakeyama
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006136:GTP Cyclohydrolase / Q000037:antagonists & inhibitors*; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D014162:Transfection
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C491065:Gchfr protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D006136:GTP Cyclohydrolase