Yoneyama T, Hatakeyama K
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
Protein Sci. 2001 Apr;10(4):871-8. doi: 10.1110/ps.38501.
GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates feedback inhibition of GTP cyclohydrolase I activity by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4), 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, BH4 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. BH4 binds to the GTP cyclohydrolase I/GFRP complex with a Kd of 4 microM, and phenylalanine binds to the protein complex with a Kd of 94 microM. The binding of BH4 is enhanced by dGTP. The binding stoichiometrics of BH4 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, BH4 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 BH4 and phenylalanine but also that each binding site of BH4 and phenylalanine may be primarily composed of residues of GTP cyclohydrolase I and GFRP, respectively.
GTP环化水解酶I反馈调节蛋白（GFRP）介导6R-L-赤藓糖型-5,6,7,8-四氢生物蝶呤（BH4）对GTP环化水解酶I活性的反馈抑制，BH4是生成儿茶酚胺、5-羟色胺和一氧化氮的关键酶以及苯丙氨酸羟化酶的必需辅因子。GFRP还在GTP水平未饱和时介导苯丙氨酸对GTP环化水解酶I活性的前馈刺激。这些配体，即BH4和苯丙氨酸，诱导一分子GTP环化水解酶I与两分子GFRP之间形成复合物。在此，我们报告使用Hummel和Dreyer的凝胶过滤法对配体结合进行的分析。BH4以4 microM的解离常数（Kd）与GTP环化水解酶I/GFRP复合物结合，苯丙氨酸以94 microM的Kd与该蛋白复合物结合。dGTP可增强BH4的结合。据估计，BH4和苯丙氨酸与每个蛋白复合物的结合化学计量比为每种10个分子，换句话说，每个蛋白亚基一个分子，因为GTP环化水解酶I是十聚体，GFRP是五聚体。平衡透析实验的数据证实了这些发现。关于配体与游离蛋白的结合，BH4与GTP环化水解酶I结合较弱，但不与GFRP结合，苯丙氨酸与GFRP结合较弱，但不与GTP环化水解酶I结合。这些结果表明，蛋白复合物的整体结构有助于BH4和苯丙氨酸的结合，但也表明BH4和苯丙氨酸的每个结合位点可能分别主要由GTP环化水解酶I和GFRP的残基组成。
