Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli.
Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli.
复制标题
大肠杆菌 GTP 环水解酶 I 的变构特征。
DOI：
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
N. Blau
中科院分区：
文献类型：
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作者：
G. Schoedon;U. Redweik;G. Frank;R. Cotton;N. Blau
使用改进的酶测定和直接测定的产品，二氢新蝶呤三磷酸的动力学和调节特性的GTP环化水解酶I进行了研究。从大肠杆菌中纯化酶至绝对均一性，如通过多达50个氨基酸残基的N-末端测序所证明的。一个30个残基的内部片段与大鼠肝GTP环化水解酶I的同源性为42%。该酶不遵循米氏动力学或显示S形反应曲线。底物饱和动力学被认为是缓慢的，低响应GTP浓度的微小变化。GTP环化水解酶I具有相对高的表观Km。用GTP-琼脂糖（100 μ M）和UTP-琼脂糖（110 μ M）纯化的酶的值略有不同。计算了相应酶制剂的12/min和19/min的低周转数。GTP-环化水解酶-I活性在Vmax中由K+、二价阳离子、UTP和四氢生物蝶呤调节。二价阳离子，如Mg 2+，具有活化作用，最佳浓度为8 mM Mg 2+。一个不同的催化功能和形成一个新的，身份不明的产品由GTP环化水解酶I观察到在Ca 2+的存在下。在1 mM EDTA和Mg ~（2+）存在下，GTP-环化水解酶-I的活性被螯合物强烈抑制。UTP被证明不是竞争性抑制剂，而是正性调节剂。UTP完全消除了螯合物的抑制作用。四氢生物蝶呤显示出抑制作用，在100 μ M四氢生物蝶呤时抑制率为50%。UTP能够减轻四氢生物蝶呤的抑制作用。使用单克隆抗体1F 11（与GTP结合位点相关）和单克隆抗体NS 7（模拟四氢生物蝶呤），证明了每个酶亚基上GTP和四氢生物蝶呤的不同结合位点。Western-blot竞争分析显示，UTP结合位点不同于GTP和四氢生物蝶呤的结合位点。基于动力学行为和所观察到的调节种类，我们将GTP环化水解酶I定义为M类变构酶。
The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP-agarose (100 microM) and UTP-agarose (110 microM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 microM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme.
果蝇中具有发育功能的代谢基因的克隆和分子表征。
影响因子：
3.3
作者：
McLean,JR;Boswell,R;O'Donnell,J
通讯作者：
O'Donnell,J
黑腹果蝇 GTP 环水解酶 I 的纯化和表征。
DOI：
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发表时间：
1986
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Weisberg,EP;O'Donnell,JM
通讯作者：
O'Donnell,JM