Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic.
J Phys Chem B. 2013 May 30;117(21):6394-402. doi: 10.1021/jp401506v. Epub 2013 May 20.
Cationic specificity which follows the Hofmeister series has been established for the catalytic efficiency of haloalkane dehalogenase LinB by a combination of molecular dynamics simulations and enzyme kinetic experiments. Simulations provided a detailed molecular picture of cation interactions with negatively charged residues on the protein surface, particularly at the tunnel mouth leading to the enzyme active site. On the basis of the binding affinities, cations were ordered as Na(+) > K(+) > Rb(+) > Cs(+). In agreement with this result, a steady-state kinetic analysis disclosed that the smaller alkali cations influence formation and productivity of enzyme-substrate complexes more efficiently than the larger ones. A subsequent systematic investigation of two LinB mutants with engineered charge in the cation-binding site revealed that the observed cation affinities are enhanced by increasing the number of negatively charged residues at the tunnel mouth, and vice versa, reduced by decreasing this number. However, the cation-specific effects are overwhelmed by strong electrostatic interactions in the former case. Interestingly, the substrate inhibition of the mutant LinB L177D in the presence of chloride salts was 7 times lower than that of LinB wild type in glycine buffer. Our work provides new insight into the mechanisms of specific cation effects on enzyme activity and suggests a potential strategy for suppression of substrate inhibition by the combination of protein and medium engineering.
阳离子特异性遵循豪夫迈斯特序列，通过分子动力学模拟和酶动力学实验的结合，确定了卤代烷脱卤酶 LinB 的催化效率。模拟提供了阳离子与蛋白质表面带负电荷的残基相互作用的详细分子图像，特别是在通向酶活性位点的隧道口处。根据结合亲和力，阳离子的顺序为 Na(+) > K(+) > Rb(+) > Cs(+)。与这一结果一致，稳态动力学分析表明，较小的碱金属阳离子比较大的碱金属阳离子更有效地影响酶-底物复合物的形成和产率。随后对阳离子结合位点进行工程化电荷的两种 LinB 突变体进行了系统研究，结果表明，观察到的阳离子亲和力通过增加隧道口处带负电荷的残基数而增强，反之亦然。然而，在前者情况下，阳离子的特异性影响被强静电相互作用所掩盖。有趣的是，在氯化物盐存在下，突变体 LinB L177D 的底物抑制作用比 Glycine 缓冲液中 LinB 野生型低 7 倍。我们的工作为特定阳离子对酶活性的影响机制提供了新的见解，并为通过蛋白质和介质工程的结合抑制底物抑制提供了潜在策略。
