Zn2+ regulation of ornithine transcarbamoylase. II. Metal binding site.
Zn2+ regulation of ornithine transcarbamoylase. II. Metal binding site.
复制标题
鸟氨酸转氨甲酰酶的 Zn2 调节。
DOI：
10.1016/0022-2836(90)90026-i
复制
发表时间：
1990
影响因子：
5.6
通讯作者：
Herzberg,W
中科院分区：
文献类型：
--
作者：
Kuo,LC;Caron,C;Lee,S;Herzberg,W
两种类型的构象变化介导的大肠杆菌鸟氨酸氨甲酰转移酶的金属离子锌。在锌的快速平衡结合后，酶经历变构转变。在没有底物的情况下，锌结合的酶进一步经历缓慢的异构化，伴随着活性损失。通过凝胶色谱和原子吸收光谱法测定，三种金属离子在异构化酶中紧密络合。由于酶是由相同的亚基组成的三聚体，每个酶单体结合一个锌离子。通过定点突变，在酶的273位的半胱氨酰残基已被鉴定为金属配体。当该残基被丙氨酸取代时，锌不再是紧密结合的抑制剂，并且不促进异构化。锌对突变酶的作用的改变归因于金属亲和力的降低。突变体酶，当饱和的金属，显示一个内在的变构不变的野生型;发现一个相同的希尔系数为1.5锌结合的Ala 273和野生型酶。Cys 273也是L-鸟氨酸的结合位点。在pH 8.5时，Ala 273酶与底物类似物L-正缬氨酸的结合弱10倍，并且表现出比野生型酶小27倍的k cat K n orn。这一发现支持了我们先前的解释，锌诱导的鸟氨酸氨甲酰转移酶的鸟氨酸协同性是由L-鸟氨酸和金属之间的直接竞争相同的网站。作为对照，细菌鸟氨酸转氨甲酰酶的剩余三个半胱氨酰残基中的每一个也被丙氨酸取代。发现这些巯基与锌络合、鸟氨酸结合或酶变构无关。
Two types of conformational changes are mediated in Escherichia coli ornithine transcarbamoylase by the metal ion zinc. Upon binding of zinc in rapid equilibrium, the enzyme undergoes an allosteric transition. In the absence of substrates, the zinc-bound enzyme further undergoes a slow isomerization with a concomitant activity loss. Three metal ions are tightly complexed in the isomerized enzyme as determined by gel chromatography and atomic absorption spectroscopy. Since the enzyme is a trimer composed of identical subunits, one zinc ion is bound per enzyme monomer. With the application of site-directed mutagenesis, the cysteinyl residue at position 273 of the enzyme has been identified as a metal ligand. When this residue is replaced by an alanine, zinc is no longer a tight-binding inhibitor and does not promote isomerization. The alteration in the action of zinc on the mutant enzyme is attributed to a reduced metal affinity. The mutant enzyme, when saturated by the metal, displays an intrinsic allostery unchanged from that of the wild-type; an identical Hill coefficient of 1.5 is found for zinc binding to the Ala273 and wild-type enzymes. Cys273 is also a binding site of l-ornithine. At pH 8.5, the Ala273 enzyme binds the substrate analog l-norvaline ten times more weakly and exhibits a k cat K n orn that is 27 times less than that of the wild-type enzyme. This finding supports our earlier interpretation that the zinc-induced ornithine co-operativity of ornithine transcarbamoylase is caused by direct competition between l-ornithine and the metal for the same site. As controls, each of the remaining three cysteinyl residues of the bacterial ornithine transcarbamoylase has also been replaced with alanine. These sulfhydryl groups are found not to be related to zinc complexation, ornithine binding or enzyme allostery.
登录
查看更多内容
影响因子：
9.8
作者：
J. Huffman
通讯作者：
J. Huffman
肠道和胰腺内分泌细胞研究中的银染色。
DOI：
--
发表时间：
1980
期刊：
Investigative & cell pathology
影响因子：
--
作者：
L. Grimelius;E. Wilander
通讯作者：
E. Wilander
兔叶状乳头味蕾的精细结构。
DOI：
10.1016/s0022-5320(67)80224-7
发表时间：
1967
期刊：
Journal of ultrastructure research
影响因子：
--
作者：
R. G. Murray;A. Murray
通讯作者：
A. Murray
影响因子：
2.1
作者：
POLLEN, JJ;DREILINGER, A
通讯作者：
DREILINGER, A
对大型 Spurr 环氧树脂切片的重新嵌入（“弹出”）区域进行诊断电子显微镜检查。
影响因子：
1
作者：
diSant'Agnese,PA;DeMesy-Jensen,KL
通讯作者：
DeMesy-Jensen,KL