Markby D W, Zhou B B, Schachman H K
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10568-72. doi: 10.1073/pnas.88.23.10568.
In an effort to clarify effects of specific protein-protein interactions on the properties of the dodecameric enzyme aspartate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), we initiated studies of a simpler complex containing an intact catalytic trimer and three copies of a fragment from the regulatory chain. The partial regulatory chain was expressed as a soluble 9-kDa zinc-binding polypeptide comprising 11 amino acids encoded by the polylinker of pUC18 fused to the amino terminus of residues 84-153 of the regulatory chain; this polypeptide includes the zinc domain detected in crystallographic studies of the holoenzyme. In contrast to intact regulatory chains, the zinc-binding polypeptide is monomeric in solution because it lacks the second domain responsible for dimer formation and assembly of the dodecameric holoenzyme. The isolated 9-kDa protein forms a tight, zinc-dependent complex with catalytic trimer, as shown by the large shift in electrophoretic mobility of the trimer in nondenaturing polyacrylamide gels. Enzyme assays of the complex showed a hyperbolic dependence of initial velocity on aspartate concentration with Vmax and Km for aspartate approximately 50% lower than the values for free catalytic subunit. A mutant catalytic subunit containing the Lys-164----Glu substitution exhibited a striking increase in enzyme activity at low aspartate concentrations upon interaction with the zinc domain because of a large reduction in Km upon complex formation. These changes in functional properties indicate that the complex of the zinc domain and catalytic trimer is an analog of the high-affinity R ("relaxed") state of aspartate transcarbamoylase, as proposed previously for a transiently formed assembly intermediate composed of one catalytic and three regulatory subunits. Conformational changes at the active sites, resulting from binding the zinc-containing polypeptide chains, were detected by difference spectroscopy with trinitrophenylated catalytic trimers. Isolation of the zinc domain of aspartate transcarbamoylase provides a model protein for study of oligomer assembly, communication between dissimilar polypeptides, and metal-binding motifs in proteins.
为了阐明特定蛋白质 - 蛋白质相互作用对十二聚体酶天冬氨酸转氨甲酰酶（氨甲酰磷酸：L - 天冬氨酸氨甲酰转移酶，EC 2.1.3.2）性质的影响，我们开始研究一种更简单的复合物，它包含一个完整的催化三聚体和来自调节链的三个片段拷贝。部分调节链被表达为一种可溶性的9 kDa锌结合多肽，其由pUC18多克隆位点编码的11个氨基酸与调节链84 - 153位残基的氨基末端融合而成；该多肽包含在全酶晶体学研究中检测到的锌结构域。与完整的调节链不同，锌结合多肽在溶液中是单体，因为它缺乏负责二聚体形成和十二聚体全酶组装的第二个结构域。分离出的9 kDa蛋白与催化三聚体形成紧密的、锌依赖性复合物，这在非变性聚丙烯酰胺凝胶中三聚体的电泳迁移率有大幅变化中得到体现。该复合物的酶活性测定表明，初始速度对天冬氨酸浓度呈双曲线依赖性，天冬氨酸的Vmax和Km值比游离催化亚基的值低约50%。含有Lys - 164→Glu替换的突变催化亚基在与锌结构域相互作用时，在低天冬氨酸浓度下酶活性显著增加，这是因为形成复合物后Km大幅降低。这些功能性质的变化表明，锌结构域与催化三聚体的复合物是天冬氨酸转氨甲酰酶高亲和力R（“松弛”）状态的类似物，正如之前针对由一个催化亚基和三个调节亚基组成的瞬时形成的组装中间体所提出的那样。通过对三硝基苯基化催化三聚体进行差示光谱法检测到，由于结合含锌多肽链导致活性位点的构象变化。天冬氨酸转氨甲酰酶锌结构域的分离为研究寡聚体组装、不同多肽之间的通讯以及蛋白质中的金属结合基序提供了一种模型蛋白。
