Cation-SpecificEffects on Enzymatic Catalysis Driven by Interactions at the Tunnel Mouth Veronika S ̌ tě pa ́ nkova ́ , †,‡ Jana Paterova ́ , § Jir ̌ í Damborsky ́ , †,‡ Pavel Jungwirth, § Radka Chaloupkova ́ ,* ,† and Jan Heyda* ,§,¶ † 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 ‡ International Clinical Research Center, St. Anne’s University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic § Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo na ́ m. 2, 166 10 Prague 6, Czech Republic * S Supporting Information ABSTRACT: 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-specificeffects 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. ■ INTRODUCTION Ion-specificeffects play an important role in biochemical and biophysical processes. Biological systems usually undergo a significant stress when salt concentrations are varied or one ion is replaced by another. 1 Traditionally, ions have been ordered in the so-called Hofmeister series according to their ability to salt out proteins. 2,3 However, many other processes, including enzymatic activity and thermal stability, also exhibit ion specificity. 4 The emerging view, based on molecular simulations and laboratory experiments, is that ion effects are actually a complex phenomenon involving local interactions between ions and functional groups at the protein surface 5 as well as ion-ion interactions in the solution. 6 Ordering cations and anions into separate series proved to be a simplification, which may not be always justified. 7,8 Nevertheless, in many cases, formulating Hofmeister series provides a reasonable starting point in attempts to understand the fundamental molecular mecha- nisms. The effects of anions on protein solubility and stability are typically stronger than those of cations, at least monovalent ones, while the enzyme activity can be significantly affected by both anions and cations. 9,10 After all, a basic asymmetry in biology which propels many physiological processes is that of the intracellular potassium versus extracellular sodium concen- trations. From the interaction point of view, these two monovalent cations differ only slightly in size, but the consequences for cellular mechanisms are dramatic. 11 At the level of enzymatic activity, the previously observed differences between effects of Na + versus K + were more subtle, but still important. 12 It was shown that replacing sodium by potassium enhances significantly the enzymatic activity of the HIV protease. 13,14 Most recently, the activity of HIV protease was studied in the presence of four alkali cations, Li + , Na + ,K + , and Cs + , and it was found out that the alkali cations can be essentially systematized into a Hofmeister-like series. 15 The likely origin of this ordering is the fact that smaller alkali cations interact more strongly than the bigger ones with the anionic carboxylic groups of the glutamate and aspartate side chains. Of particular importance could be a pair of aspartates at the entrance to the active site where more strongly bound cations may hamper the binding of a mostly hydrophobic substrate. 14,15 The strength of this effect is, however, not yet unequivocally established. 16 Received: February 11, 2013 Revised: April 23, 2013 Published: April 29, 2013 Article pubs.acs.org/JPCB © 2013 American Chemical Society 6394 dx.doi.org/10.1021/jp401506v | J. Phys. Chem. B 2013, 117, 6394-6402