The Effect of Osmolytes on Protein Dynamics in the LDH- Catalyzed

Laser induced temperature jump relaxation spectroscopy was used to probe the effect of osmolytes on the microscopic rate constants of the LDH-catalyzed reaction. NADH fluorescence and absorption relaxation kinetics were measured for the LDH reaction system in presence of varying amounts of trimethylamine-N-oxide (TMAO), a protein stabilizing osmolyte, or urea, a protein destabilizing osmolyte. TMAO in 1 M concentration strongly increases the rate of hydride transfer, nearly nullifies its activation energy, and also slightly increases the enthalpy of hydride transfer. In 1 M urea the hydride transfer enthalpy is almost nullified, but activation energy of the step is not affected significantly. TMAO increases preference of closed conformation of the active site loop in LDH•NAD+•lactate complex; urea decreases it. The loop-opening rate in LDH•NADH•pyruvate complex changes its temperature dependence to inverse-Arrhenius with TMAO. In this complex, urea speeds up the loop motion, without changing the loop opening enthalpy. A strong, non-Arrhenius drop in pyruvate binding rate in presence of TMAO offers a decrease in the fraction of the open-loop, pyruvate-binding competent form at higher temperatures. Pyruvate off-rate is not affected by urea, but decreases with TMAO. Thus, the osmolytes strongl