Eberhard M, Tsai-Pflugfelder M, Bolewska K, Hommel U, Kirschner K
Abteilung Biophysikalische Chemie, Biozentrum der Universität Basel, Switzerland.
Biochemistry. 1995 Apr 25;34(16):5419-28. doi: 10.1021/bi00016a013.
Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.
通过在基因水平上分离结构域并将其表达为单功能蛋白，对来自大肠杆菌的单体双功能酶吲哚甘油磷酸合酶：磷酸核糖基邻氨基苯甲酸异构酶的假定结构域-结构域相互作用进行了探究。发现工程化的单功能酶是稳定的单体蛋白，具有几乎完全的催化活性。此外，吲哚甘油磷酸与吲哚甘油磷酸合酶活性位点的结合以及还原型1-[(2-羧基苯基)氨基]-1-脱氧核糖-5-磷酸（吲哚甘油磷酸合酶和磷酸核糖基邻氨基苯甲酸异构酶的竞争性抑制剂）的结合在单功能和双功能酶中几乎相同。此外，无论是通过沉降和凝胶过滤实验在体外，还是通过在同一细胞中共表达结构域在体内，都未发现单功能酶之间存在关联。因此，在功能水平上未发现大肠杆菌的双功能酶相对于各自的单功能酶具有选择性优势。然而，磷酸核糖基邻氨基苯甲酸异构酶结构域似乎通过似乎微妙影响配体结合动力学的相互作用来稳定大肠杆菌双功能酶的吲哚甘油磷酸合酶结构域。
