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A platform for chemical modification of mandelate racemase: characterization of the C92S/C264S and γ-thialysine 166 variants.
Protein Engineering, Design and Selection ( IF 2.6 ) Pub Date : 2018-06-01 , DOI: 10.1093/protein/gzy011
Mitesh Nagar 1 , Himank Kumar 1 , Stephen L Bearne 1, 2
Affiliation  

Mandelate racemase (MR) serves as a paradigm for our understanding of enzyme-catalyzed deprotonation of a carbon acid substrate. To facilitate structure-function studies on MR using non-natural amino acid substitutions, we engineered the Cys92Ser/Cys264Ser variant (dmMR) as a platform for introducing Cys residues at specific locations for subsequent covalent modification. While the highly reactive thiol of Cys furnishes a site for chemical modification, site-specificity requires that other Cys residues be non-reactive or replaced by a non-reactive amino acid, especially if chemical modification is conducted under denaturing conditions. The catalytic efficiency of dmMR is reduced only ~2-fold relative to wild-type MR, making dmMR a viable platform for the site-specific introduction of Cys. As an example, the inactive Lys166Cys variant of dmMR was treated with ethylenimine under denaturing conditions to replace the Brønsted acid-base catalyst Lys 166 with the non-natural amino acid γ-thialysine. Comparison of the pH-activity profiles of dmMR and the active γ-thialysine variant revealed a reduction in the pKa for the side chain amino group of ~0.4 units for the latter variant. Unlike wild-type MR for which diffusion is partially rate-limiting, dmMR and the γ-thialysine variant showed no dependence on the solvent viscosity suggesting that the chemical step is fully rate-limiting.

中文翻译:

化学修饰扁桃酸酯消旋酶的平台:表征C92S / C264S和γ-硫代赖氨酸166变体。

扁桃酸酯消旋酶(MR)充当了我们对碳酸底物的酶催化去质子化理解的范例。为了促进使用非天然氨基酸取代的MR的结构功能研究,我们设计了Cys92Ser / Cys264Ser变体(dmMR)作为在特定位置引入Cys残基进行后续共价修饰的平台。虽然Cys的高反应性硫醇提供了一个化学修饰位点,但位点特异性要求其他Cys残基不具有反应性或被非反应性氨基酸取代,尤其是在变性条件下进行化学修饰的情况下。相对于野生型MR,dmMR的催化效率仅降低约2倍,从而使dmMR成为位点特异性导入Cys的可行平台。举个例子,dmMR的非活性Lys166Cys变体在变性条件下用亚乙基亚胺处理,以用非天然氨基酸γ-硫代赖氨酸替代布朗斯台德酸碱催化剂Lys 166。比较dmMR和活性γ-硫赖氨酸变体的pH活性曲线,发现后一种变体的侧链氨基的pKa降低了约0.4个单位。与野生型MR的扩散部分受到限速作用不同,dmMR和γ-硫赖氨酸变体显示出对溶剂粘度的依赖性,这表明化学步骤是完全限速的。比较dmMR和活性γ-硫赖氨酸变体的pH活性曲线,发现后一种变体的侧链氨基的pKa降低了约0.4个单位。与野生型MR的扩散部分受到限速作用不同,dmMR和γ-硫赖氨酸变体显示出对溶剂粘度的依赖性,这表明化学步骤是完全限速的。比较dmMR和活性γ-硫赖氨酸变体的pH活性曲线,发现后一种变体的侧链氨基的pKa降低了约0.4个单位。与野生型MR的扩散部分受到限速作用不同,dmMR和γ-硫赖氨酸变体显示出对溶剂粘度的依赖性,这表明化学步骤是完全限速的。
更新日期:2019-11-01
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