Murata L B, Schachman H K
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Protein Sci. 1996 Apr;5(4):709-18. doi: 10.1002/pro.5560050416.
Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site-directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C-terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C-terminal helix. Truncations and substitutions within the predicted C-terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar.
当同源蛋白质的结构已知时，从氨基酸序列预测蛋白质的三级结构会得到极大的便利。在此基础上，基于大肠杆菌天冬氨酸转氨甲酰酶（ATCase）催化（c）链的已知三级结构，通过定点诱变实验研究了大肠杆菌鸟氨酸转氨甲酰酶（OTCase）的结构特征。在ATCase中，每条c链由两个球状结构域组成，这两个结构域由两个结构域间的螺旋相连，其中一个螺旋靠近C端，对链在体内的折叠及其组装成三聚体至关重要。每个活性位点位于两条链之间的界面处，需要相邻每条链上的残基参与。OTCase是一种三聚体酶，基于序列同一性（32%）、该酶催化反应的性质以及二级结构预测，有人提出它在结构上与ATCase三聚体相似。如下所示，对OTCase和ATCase序列的分析揭示了在与ATCase活性位点和C端螺旋相对应的部分存在广泛的进化保守性。OTCase预测的C端螺旋内的截短和替换对活性和热稳定性的影响与ATCase中类似改变所引起的影响惊人地相似。同样，OTCase假定活性位点中两个保守残基Ser 55和Lys 86中的任何一个发生替换，都会产生与ATCase类似替换所引起的有害影响相似的有害影响。由这两个相对无活性的OTCase突变体的链组成的杂合三聚体表现出显著增加的活性，正如位于链界面处的共享活性位点所预测的那样。这些结果有力地支持了这两种酶的三级和四级结构相似的假说。
