Sims Paul A, Menefee Ann L, Larsen Todd M, Mansoorabadi Steven O, Reed George H
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53726, USA.
J Mol Biol. 2006 Jan 20;355(3):422-31. doi: 10.1016/j.jmb.2005.10.050. Epub 2005 Nov 8.
Enolase is a dimeric enzyme that catalyzes the interconversion of 2-phospho-D-glycerate and phosphoenolpyruvate. This reversible dehydration is effected by general acid-base catalysis that involves, principally, Lys345 and Glu211 (numbering system of enolase 1 from yeast). The crystal structure of the inactive E211Q enolase shows that the protein is properly folded. However, K345 variants have, thus far, failed to crystallize. This problem was solved by crystallization of an engineered heterodimer of enolase. The heterodimer was composed of an inactive subunit that has a K345A mutation and an active subunit that has N80D and N126D surface mutations to facilitate ion-exchange chromatographic separation of the three dimeric species. The structure of this heterodimeric variant, in complex with substrate/product, was obtained at 1.85 A resolution. The structure was compared to a new structure of wild-type enolase obtained from crystals belonging to the same space group. Asymmetric dimers having one subunit exhibiting two of the three active site loops in an open conformation and the other in a conformation having all three loops closed appear in both structures. The K345A subunit of the heterodimer is in the loop-closed conformation; its Calpha carbon atoms closely match those of the corresponding subunit of wild-type enolase (root-mean-squared deviation of 0.23 A). The kcat and kcat/Km values of the heterodimer are approximately half those of the N80D/N126D homodimer, which suggests that the subunits in solution are kinetically independent. A comparison of enolase structures obtained from crystals belonging to different space groups suggests that asymmetric dimers can be a consequence of the asymmetric positioning of the subunits within the crystal lattice.
烯醇化酶是一种二聚体酶，催化2-磷酸-D-甘油酸和磷酸烯醇丙酮酸之间的相互转化。这种可逆的脱水反应通过一般酸碱催化实现，主要涉及赖氨酸345（来自酵母烯醇化酶1的编号系统）和谷氨酸211。无活性的E211Q烯醇化酶的晶体结构表明该蛋白质折叠正确。然而，到目前为止，K345变体未能结晶。通过烯醇化酶工程异二聚体的结晶解决了这个问题。该异二聚体由一个具有K345A突变的无活性亚基和一个具有N80D和N126D表面突变的活性亚基组成，以促进三种二聚体物种的离子交换色谱分离。该异二聚体变体与底物/产物复合物的结构在1.85 Å分辨率下获得。将该结构与从属于同一空间群的晶体中获得的野生型烯醇化酶的新结构进行比较。在两种结构中都出现了不对称二聚体，其中一个亚基的三个活性位点环中的两个处于开放构象，另一个亚基的三个环都处于闭合构象。异二聚体的K345A亚基处于环闭合构象；其α碳原子与野生型烯醇化酶相应亚基的α碳原子紧密匹配（均方根偏差为0.23 Å）。异二聚体的kcat和kcat/Km值约为N80D/N126D同二聚体的一半，这表明溶液中的亚基在动力学上是独立的。从属于不同空间群的晶体中获得的烯醇化酶结构的比较表明，不对称二聚体可能是亚基在晶格中不对称定位的结果。
