Unveiling the influence of salt concentration on the different stages of the catalytic cycle of a halophilic enzyme

Enzymes from halophilic organisms function in near-saturating salt concentrations. To explore enzyme adaptation to these conditions and the broader effects of salt on enzyme catalysis, we examined the salt effect on the kinetics of a glucose-6-phosphate dehydrogenase from the halophilic archaeon Haloferax volcanii (HvG6PDH). This enzyme has a negatively charged surface and catalyzes the oxidation of the negatively charged substrate glucose-6-phosphate (G6PDH activity) and glucose (glcDH activity). We found strikingly different salt effects on the two activities. Steady-state kinetics experiments revealed a 50-fold decrease in K_M ^G6P for G6PDH activity. Instead, for glcDH activity, the main effects were a 10-fold increase in k_cat and a 10-fold increase in k_cat ^glc/K_M ^glc. The effect of salt in each step of the catalytic cycle was assessed using pre-steady-state kinetic experiments. We found that KCl increases both the binding and release rates of NAD⁺ in G6PDH activity, whereas in glcDH it does not affect binding but decreases the release rate. In G6PDH, KCl does not affect the chemical step, while in glcDH this step is significantly accelerated. The results further indicate that the increase in G6P affinity is the main contributor to the increase in G6PDH activity, whereas the increase in glcDH activity is primarily driven by the increase in the chemical step rate. Together, these findings highlight charge screening as a key factor underlying the differential effects of salt, by reducing electrostatic repulsion between the enzyme surface and its substrates and modulating interactions between the positively charged active site and the substrates.