ABSTRACT
Recent studies exploring the relationship between enzymatic catalysis and protein dynamics in the aqueous phase have yielded evidence that dynamics and enzyme activity are strongly correlated. Given that protein dynamics are significantly attenuated in organic solvents and that proteins exhibit a wide range of motions depending on the specific solvent environment, the nonaqueous milieu provides a unique opportunity to examine the role of protein dynamics in enzyme activity. Variable-temperature kinetic measurements, X-band electron spin resonance spectroscopy, (1)H NMR relaxation, and (19)F NMR spectroscopy experiments were performed on subtilisin Carlsberg colyophilized with several inorganic salts and suspended in organic solvents. The results indicate that salt activation induces a greater degree of transition-state flexibility, reflected by a more positive DeltaDeltaS(dagger), for the more active biocatalyst preparations in organic solvents. In contrast, DeltaDeltaH(dagger) was negligible regardless of salt type or salt content. Electron spin resonance spectroscopy and (1)H NMR relaxation measurements, including spin-lattice relaxation, spin-lattice relaxation in the rotating frame, and longitudinal magnetization exchange, revealed that the enzyme's turnover number (k(cat)) was strongly correlated with protein motions in the centisecond time regime, weakly correlated with protein motions in the millisecond regime, and uncorrelated with protein motions on the piconanosecond timescale. In addition, (19)F chemical shift measurements and hyperfine tensor measurements of biocatalyst formulations inhibited with 4-fluorobenzenesulfonyl fluoride and 4-ethoxyfluorophosphinyl-oxy-TEMPO, respectively, suggest that enzyme activation was only weakly affected by changes in active-site polarity.
