Murphy Paul M, Bolduc Jill M, Gallaher Jasmine L, Stoddard Barry L, Baker David
Department of Biochemistry, Medical Scientist Training Program, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2009 Jun 9;106(23):9215-20. doi: 10.1073/pnas.0811070106. Epub 2009 May 21.
Altering the specificity of an enzyme requires precise positioning of side-chain functional groups that interact with the modified groups of the new substrate. This requires not only sequence changes that introduce the new functional groups but also sequence changes that remodel the structure of the protein backbone so that the functional groups are properly positioned. We describe a computational design method for introducing specific enzyme-substrate interactions by directed remodeling of loops near the active site. Benchmark tests on 8 native protein-ligand complexes show that the method can recover native loop lengths and, often, native loop conformations. We then use the method to redesign a critical loop in human guanine deaminase such that a key side-chain interaction is made with the substrate ammelide. The redesigned enzyme is 100-fold more active on ammelide and 2.5e4-fold less active on guanine than wild-type enzyme: The net change in specificity is 2.5e6-fold. The structure of the designed protein was confirmed by X-ray crystallographic analysis: The remodeled loop adopts a conformation that is within 1-A Calpha RMSD of the computational model.
改变酶的特异性需要侧链官能团精确排列，这些官能团与新底物的修饰基团相互作用。这不仅需要引入新官能团的序列变化，还需要重塑蛋白质主链结构的序列变化，以便官能团正确定位。我们描述了一种计算设计方法，通过对活性位点附近的环进行定向重塑来引入特定的酶-底物相互作用。对8种天然蛋白质-配体复合物的基准测试表明，该方法可以恢复天然环长度，并且通常能恢复天然环构象。然后我们使用该方法重新设计人鸟嘌呤脱氨酶中的一个关键环，使其与底物氨甲酰脲形成关键的侧链相互作用。重新设计的酶对氨甲酰脲的活性比野生型酶高100倍，对鸟嘌呤的活性比野生型酶低2.5×10⁴倍：特异性的净变化为2.5×10⁶倍。通过X射线晶体学分析证实了设计蛋白质的结构：重塑的环采用的构象与计算模型的α碳原子均方根偏差在1埃以内。
