Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase.
Regeneration of active enzyme by formation of hybrids from inactive derivatives: implications for active sites shared between polypeptide chains of aspartate transcarbamoylase.
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通过从无活性衍生物形成杂合体来再生活性酶：对天冬氨酸转氨甲酰酶多肽链之间共享活性位点的影响。
DOI：
10.1073/pnas.82.2.361
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发表时间：
1985
影响因子：
11.1
通讯作者：
Schachman,HK
中科院分区：
文献类型：
--
作者：
Robey,EA;Schachman,HK
大肠杆菌天冬氨酸氨甲酰转移酶（天冬氨酸氨甲酰转移酶，EC 2.1.3.2）的晶体学研究结合化学修饰实验已经导致这样的建议，即酶的活性位点在催化三聚体的相邻多肽链之间的界面处，并且涉及来自相邻链的氨基酸残基的联合参与。然而，活性位点和参与催化的残基的精确位置尚不清楚。为了检验链之间共享活性位点的假设，我们构建了混合三聚体，其中两条链在一个假定的活性位点残基处被修饰，第三条链在不同的活性位点残基处被改变。一个亲本三聚体是还原的磷酸吡哆醛衍生物，其中赖氨酸-84被修饰，另一个是突变蛋白，其中酪氨酸-165通过定点诱变转化为丝氨酸。在促进链间交换的条件下孵育这两种几乎无活性的衍生物的混合物导致酶活性的大幅增加，大约对应于每个三聚体形成一个活性位点。纯化的杂合三聚体，含有两个吡啶氧基化和一个突变链或反之亦然，分别具有23%和28%，天然野生型催化三聚体的活性，相比之下，5%和3%的亲本三聚体。活性大幅增加的最可能的解释是每个杂交三聚体中形成了一个“天然”活性位点。这些结果构成了强有力的证据，共享的活性位点的天冬氨酸氨基甲酸转移酶。
Crystallographic studies of Escherichia coli aspartate transcarbamoylase (aspartate carbamoyltransferase, EC 2.1.3.2) in conjunction with chemical modification experiments have led to the suggestion that the active sites of the enzyme are at the interfaces between adjacent polypeptide chains of the catalytic trimers and involve joint participation of amino acid residues from the adjoining chains. However, the precise locations of the active sites and of the residues involved in catalysis are not known. To test the hypothesis that the active sites are shared between chains, we constructed hybrid trimers in which two chains were modified at one presumed active site residue and the third chain was altered at a different active site residue. One parental trimer was a reduced pyridoxal phosphate derivative in which lysine-84 was modified and the other was a mutant protein in which tyrosine-165 was converted to serine by site-directed mutagenesis. Incubating mixtures of these two virtually inactive derivatives under conditions promoting interchain exchange led to a large increase in enzyme activity corresponding approximately to the formation of one active site per trimer. The purified hybrid trimers, containing either two pyridoxylated and one mutant chain or vice versa, had 23% and 28%, respectively, the activity of native wild-type catalytic trimers, compared to 5% and 3% for the parental trimers. The most likely explanation for this large increase in activity is the formation of one "native" active site in each of the hybrid trimers. The results constitute strong evidence for shared active sites in aspartate transcarbamoylase.