Active-site motions and polarity enhance catalytic turnover of hydrated subtilisin dissolved in organic solvents.

The enzyme subtilisin Carlsberg was surfactant-solubilized into two organic solvents, isooctane and tetrahydrofuran, and hydrated through stepwise changes in the thermodynamic water activity, a(w). The apparent turnover number k(cat)(app) in these systems ranged from 0.2 to 80 s(-1) and increased 11-fold in isooctane and up to 50-fold in tetrahydrofuran with increasing a(w). (19)F NMR relaxation experiments employing an active-site inhibitor were used to assess the dependence of active-site motions on a(w). The rates of NMR-derived fast (k > 10(7) s(-1)) and slow (k < 10(4) s(-1)) active-site motions increased in both solvents upon hydration, but only the slow motions correlated with k(cat). The (19)F chemical shift was a sensitive probe of the local electronic environment and provided an empirical measure of the active-site dielectric constant epsilon(as), which increased with hydration to epsilon(as) approximately 13 in each solvent. In both solvents, the transition state free energy data and epsilon(as) followed Kirkwood's model for the continuum solvation of a dipole, indicating that water also enhanced catalysis by altering the active-site's electronic environment and increasing its polarity to better stabilize the transition state. These results reveal that favorable dynamic and electrostatic effects both contribute to accelerated catalysis by solubilized subtilisin Carlsberg upon hydration in organic solvents.

Biocatalyst activity in nonaqueous environments correlates with centisecond-range protein motions.

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.

Water dynamics and salt-activation of enzymes in organic media: mechanistic implications revealed by NMR spectroscopy.

Deuterium spin relaxation was used to examine the motion of enzyme-bound water on subtilisin Carlsberg co-lyophilized with inorganic salts for activation in different organic solvents. Spectral editing was used to ensure that the relaxation times were associated with relatively mobile deuterons, which were contributed almost entirely by D(2)O rather than hydrogen-deuteron exchange on the protein. The results indicate that the timescale of motion for residual water molecules on the biocatalyst, (tau(c))(D(2)O), in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% CsF) as (k(cat)/K(M))(app) of the biocatalyst preparation increased from 0.092 s(-1) x M(-1) (salt-free) to 1,140 s(-1) x M(-1) (98% CsF). A similar effect was apparent in acetone; the timescale decreased from 24 ns (salt-free) to 2.87 ns (98% KF), with a corresponding increase in (k(cat)/K(M))(app) of 0.140 s(-1) x M(-1) (salt-free) to 12.8 s(-1) x M(-1) (98% KF). Although a global correlation between water mobility and enzyme activity was not evident, linear correlations between ln[(k(cat)/K(M))(app)] and (tau(c))(D(2)O) were obtained for salt-activated enzyme preparations in both hexane and acetone. Furthermore, a direct correlation was evident between (k(cat)/K(M))(app) and the total amount of mobile water per mass of enzyme. These results suggest that increases in enzyme-bound water mobility mediated by the presence of salt act as a molecular lubricant and enhance enzyme flexibility in a manner functionally similar to temperature. Greater flexibility may permit a larger degree of local transition-state mobility, reflected by a more positive entropy of activation, for the salt-activated enzyme compared with the salt-free enzyme. This increased mobility may contribute to the dramatic increases in biocatalyst activity.

Biocatalysis in semi-aqueous and nearly anhydrous conditions.

In the past few years there have been prolific advances in activating enzymes for nonaqueous biocatalysis. Molecular dynamics simulations complement recent experimental results and offer new insights into the deleterious effects of organic solvents, such as water stripping and active-site penetration. Methods for activating enzymes in semi-aqueous or nonaqueous media include protein engineering, chemical modification, and co-lyophilization with non-buffer salts. Enzyme immobilization on novel polymeric supports and the use of zeolite molecular sieves can also increase solvent tolerance, enhance activity, and improve enantioselectivity. The recent implementation of enzymes in ionic liquids has also led to better long-term stability relative to traditional organic solvents and the simultaneous solubilization of enzymes, cofactors and substrates.