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 feedb ack inhibition of GTP cyclohydrolase I activity by tetrahydrobiopterin and also mediates the stimulatory effect of phenylalanine on the enzy me activity. To characterize the molecular structure of GFRP, we have purified it from rat liver using an efficient step of affinity chromat ography and isolated cDNA clones, based on partial amino acid sequence s of peptides derived from purified GFRP, Comparison between the amino acid sequence deduced from the cDNA and the N-terminal amino acid seq uence of purified GFRP showed that the mature form of GFRP consists of 83 amino acid residues with a calculated M(r) of 9,542. The isolated GFRP cDNA was expressed in Escherichia coli as a fusion protein with s ix consecutive histidine residues at its N terminus. The fusion protei n was affinity-purified and digested with thrombin to remove the histi dine tag, The resulting recombinant GFRP showed kinetic properties sim ilar to those of GFRP purified from rat liver, Cross Linking experimen ts using dimethyl suberimidate indicated that GFRP was a pentamer of 5 2 kDa. Sedimentation equilibrium measurements confirmed the pentameric structure of GFRP by giving an average M(r) of 49,734, which is 5 tim es 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 CTP cyclohydrolase I complexe s.