Dutta Sneha, Moitra Anasuya, Mukherjee Debanjan, Jarori Gotam K
Department of Biological Sciences Tata Institute of Fundamental Research Mumbai India.
Present address: T. H. Chan School of Public Health Graduate School of Arts and Sciences Harvard University Boston MA USA.
FEBS Open Bio. 2017 Jun 5;7(7):892-904. doi: 10.1002/2211-5463.12242. eCollection 2017 Jul.
enolase (Pfeno) is a dimeric enzyme with multiple moonlighting functions. This enzyme is thus a potential target for anti-malarial treatments. A unique feature of Pfeno is the presence of a pentapeptide insert EWGWS . The functional role of tryptophan residues in this insert was investigated using site-directed mutagenesis. Replacement of these two Trp residues with alanines (or lysines) resulted in a near complete loss of enolase activity and dissociation of the normal dimeric form into monomers. Molecular modeling indicated that R forms π-cation bonds with the aromatic rings of W and Y. Mutation induced changes in the interactions among these three residues were presumably relayed to the inter-subunit interface via a coil formed by Y : Y, resulting in the disruption of a salt bridge between R : E and a π-cation interaction between R : Y. This led to a drop of ~ 4 kcal·mole in the inter-subunit docking energy in the mutant, causing a ~ 10 fold decrease in affinity. Partial restoration of the inter-subunit interactions led to reformation of dimers and also restored a significant fraction of the lost enzyme activity. These results suggested that the perturbations in the conformation of the surface loop containing the insert sequence were relayed to the interface region, causing dimer dissociation that, in turn, disrupted the enzyme's active site. Since enolase is a moonlighting protein with multiple parasite-specific functions, it is likely that these functions may map on to the highly conserved unique insert region of this protein.
Enolase(EC4.2.1.11).
烯醇化酶（Pfeno）是一种具有多种兼职功能的二聚体酶。因此，这种酶是抗疟疾治疗的潜在靶点。Pfeno的一个独特特征是存在一个五肽插入序列EWGWS。使用定点诱变研究了该插入序列中色氨酸残基的功能作用。用丙氨酸（或赖氨酸）取代这两个色氨酸残基导致烯醇化酶活性几乎完全丧失，正常二聚体形式解离为单体。分子建模表明，R与W和Y的芳香环形成π-阳离子键。突变引起的这三个残基之间相互作用的变化可能通过由Y:Y形成的线圈传递到亚基间界面，导致R:E之间的盐桥和R:Y之间的π-阳离子相互作用被破坏。这导致突变体中亚基对接能量下降约4千卡·摩尔，亲和力下降约10倍。亚基间相互作用的部分恢复导致二聚体重组，也恢复了很大一部分丧失的酶活性。这些结果表明，包含插入序列的表面环构象的扰动被传递到界面区域，导致二聚体解离，进而破坏酶的活性位点。由于烯醇化酶是一种具有多种寄生虫特异性功能的兼职蛋白，这些功能可能映射到该蛋白高度保守的独特插入区域。
烯醇化酶（EC4.2.1.11）。
