Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme.
Purification and characterization of rat liver GTP cyclohydrolase I. Cooperative binding of GTP to the enzyme.
复制标题
大鼠肝脏 GTP 环化水解酶 I 的纯化和表征。GTP 与酶的协同结合。
DOI：
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发表时间：
1989
影响因子：
4.8
通讯作者：
Hiroyuki Kagamiyamay
中科院分区：
文献类型：
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作者：
Kazuyuki Hatakeyama;Toshie Harada;Shuhei SuzukiS;Yasuyoshi Watanabeg;Hiroyuki Kagamiyamay
GTP环化水解酶I是催化四氢生物蝶呤生物合成途径中第一步的酶，已从大鼠肝脏提取物中纯化约38，000倍至表观均一性，产率为5%。通过Ultrogel AcA 34上的凝胶过滤，估计酶的分子量为300，000。纯化的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上在对应于分子量为30，000的位置处产生单一条带。N-末端氨基酸序列分析在Edman降解的每个步骤得到单个氨基酸，直至残基10。这些结果表明，该酶可能是一种均聚物。该酶在底物（GTP）浓度为10-50 μ M时表现出正协同性，希尔系数为2.4。酶的Vmax值为45 nmol/min.mg蛋白质。在KCl浓度为0.1 M时，产生半最大速度的GTP浓度为30 μ M。该值随着KCl浓度的升高而增加，而Vmax或Hill数没有任何变化。四氢生物蝶呤的生物合成可能受细胞内GTP水平的控制。
GTP cyclohydrolase I, an enzyme that catalyzes the first step in the biosynthetic pathway of tetrahydrobiopterin, has been purified about 38,000-fold to apparent homogeneity from rat liver extract with a yield of 5%. The molecular weight of the enzyme was estimated to be 300,000 by gel filtration on Ultrogel AcA 34. The purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position corresponding to a molecular weight of 30,000. N-terminal amino acid sequence analysis gave a single amino acid at every step of the Edman degradation up to residue 10. These results suggest that the enzyme is probably a homopolymer. The enzyme showed positive cooperativity with a Hill coefficient of 2.4 at a substrate (GTP) concentration of 10-50 microM. The Vmax value of the enzyme was 45 nmol/min.mg protein. The GTP concentration producing half-maximal velocity was 30 microM at a KCl concentration of 0.1 M. This value increased as the KCl concentration rose, without any change in Vmax or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.