Sträter N, Schnappauf G, Braus G, Lipscomb W N
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
Structure. 1997 Nov 15;5(11):1437-52. doi: 10.1016/s0969-2126(97)00294-3.
Chorismate mutase (CM) catalyzes the Claisen rearrangement of chorismate to prephenate, notably the only known enzymatically catalyzed pericyclic reaction in primary metabolism. Structures of the enzyme in complex with an endo-oxabicyclic transition state analogue inhibitor, previously determined for Bacillus subtilis and Escherichia coli CM, provide structural insight into the enzyme mechanism. In contrast to these bacterial CMs, yeast CM is allosterically regulated in two ways: activation by tryptophan and inhibition by tyrosine. Yeast CM exists in two allosteric states, R (active) and t (inactive).
We have determined crystal structures of wild-type yeast CM cocrystallized with tryptophan and an endo-oxabicyclic transition state analogue inhibitor, of wild-type yeast CM co-crystallized with tyrosine and the endo-oxabicyclic transition state analogue inhibitor and of the Thr226-->Ser mutant of yeast CM in complex with tryptophan. Binding of the transition state analogue inhibitor to CM keeps the enzyme in a 'super R' state, even if the inhibitory effector tyrosine is bound to the regulatory site.
The endo-oxabicyclic inhibitor binds to yeast CM in a similar way as it does to the distantly related CM from E. coli. The inhibitor-binding mode supports a mechanism by which polar sidechains of the enzyme bind the substrate in the pseudo-diaxial conformation, which is required for catalytic turnover. A lysine and a protonated glutamate sidechain have a critical role in the stabilization of the transition state of the pericyclic reaction. The allosteric transition from T-->R state is accompanied by a 15 degrees rotation of one of the two subunits relative to the other (where 0 degrees rotation defines the T state). This rotation causes conformational changes at the dimer interface which are transmitted to the active site. An allosteric pathway is proposed to include residues Phe28, Asp24 and Glu23, which move toward the activesite cavity in the T state. In the presence of the transition-state analogue a super R state is formed, which is characterised by a 22 degrees rotation of one subunit relative to the other.
分支酸变位酶（CM）催化分支酸的克莱森重排反应生成预苯酸，这是初级代谢中唯一已知的酶促催化周环反应。先前已测定了与内型氧杂双环过渡态类似物抑制剂结合的枯草芽孢杆菌和大肠杆菌CM的结构，这些结构为该酶的作用机制提供了结构上的见解。与这些细菌CM不同，酵母CM受到两种方式的变构调节：色氨酸激活和酪氨酸抑制。酵母CM存在两种变构状态，即R（活性）态和T（非活性）态。
我们测定了与色氨酸和内型氧杂双环过渡态类似物抑制剂共结晶的野生型酵母CM、与酪氨酸和内型氧杂双环过渡态类似物抑制剂共结晶的野生型酵母CM以及与色氨酸结合的酵母CM的Thr226→Ser突变体的晶体结构。即使抑制效应物酪氨酸结合到调节位点，过渡态类似物抑制剂与CM的结合也会使酶保持在“超R”状态。
内型氧杂双环抑制剂与酵母CM的结合方式与其与大肠杆菌中亲缘关系较远的CM的结合方式相似。抑制剂的结合模式支持一种机制，即酶的极性侧链以假双轴构象结合底物，这是催化周转所必需的。赖氨酸和质子化的谷氨酸侧链在周环反应过渡态的稳定中起关键作用。从T态到R态的变构转变伴随着两个亚基之一相对于另一个亚基15度的旋转（其中0度旋转定义T态）。这种旋转导致二聚体界面处的构象变化，并传递到活性位点。提出了一条变构途径，包括在T态向活性位点腔移动的Phe28、Asp24和Glu23残基。在存在过渡态类似物的情况下，会形成超R态，其特征是一个亚基相对于另一个亚基旋转22度。
