Rhee S, Parris K D, Hyde C C, Ahmed S A, Miles E W, Davies D R
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Jun 24;36(25):7664-80. doi: 10.1021/bi9700429.
Three-dimensional structures are reported for a mutant (betaK87T) tryptophan synthase alpha2beta2 complex with either the substrate L-serine (betaK87T-Ser) or product L-tryptophan (betaK87T-Trp) at the active site of the beta-subunit, in which both amino acids form external aldimines with the coenzyme, pyridoxal phosphate. We also present structures with L-serine bound to the beta site and either alpha-glycerol 3-phosphate (betaK87T-Ser-GP) or indole-3-propanol phosphate (betaK87T-Ser-IPP) bound to the active site of the alpha-subunit. The results further identify the substrate and product binding sites in each subunit and provide insight into conformational changes that occur upon formation of these complexes. The two structures having ligands at the active sites of both alpha- and beta-subunits reveal an important new feature, the ordering of alpha-subunit loop 6 (residues 179-187). Closure of loop 6 isolates the active site of the alpha-subunit from solvent and results in interaction between alphaThr183 and the catalytic residue alphaAsp60. Other conformational differences between the wild type and these two mutant structures include a rigid-body rotation of the alpha-subunit of approximately 5 degrees relative to the beta-subunit and large movements of part of the beta-subunit (residues 93-189) toward the rest of the beta-subunit. Much smaller differences are observed in the betaK87T-Ser structure. Remarkably, binding of tryptophan to the beta active site results in conformational changes very similar to those observed in the betaK87T-Ser-GP and betaK87T-Ser-IPP structures, with exception of the disordered alpha-subunit loop 6. These large-scale changes, the closure of loop 6, and the movements of a small number of side chains in the alpha-beta interaction site provide a structural base for interpreting the allosteric properties of tryptophan synthase.
报道了一种突变型（βK87T）色氨酸合成酶α2β2复合物的三维结构，该复合物在β亚基的活性位点分别结合有底物L-丝氨酸（βK87T-Ser）或产物L-色氨酸（βK87T-Trp），其中这两种氨基酸均与辅酶磷酸吡哆醛形成外部醛亚胺。我们还展示了L-丝氨酸结合到β位点且α-甘油3-磷酸（βK87T-Ser-GP）或吲哚-3-丙醇磷酸（βK87T-Ser-IPP）结合到α亚基活性位点的结构。这些结果进一步确定了每个亚基中的底物和产物结合位点，并深入了解了这些复合物形成时发生的构象变化。α和β亚基活性位点均有配体的这两种结构揭示了一个重要的新特征，即α亚基环6（第179 - 187位残基）的有序化。环6的闭合将α亚基的活性位点与溶剂隔离，并导致αThr183与催化残基αAsp60之间发生相互作用。野生型与这两种突变结构之间的其他构象差异包括α亚基相对于β亚基大约5度的刚体旋转以及β亚基的一部分（第93 - 189位残基）向β亚基其余部分的大幅移动。在βK87T-Ser结构中观察到的差异要小得多。值得注意的是，色氨酸与β活性位点的结合导致的构象变化与在βK87T-Ser-GP和βK87T-Ser-IPP结构中观察到的非常相似，除了α亚基环6无序。这些大规模变化、环6的闭合以及α-β相互作用位点中少数侧链的移动为解释色氨酸合成酶的变构性质提供了结构基础。
