Salt activation of enzymes in organic solvents: Mechanistic insights revealed through magnetic resonance spectroscopy

Salt-activation of proteolytic enzymes in organic solvents has been particularly well studied and optimized (e.g., with respect to water content, pH, lyophilization time, salt content, Jones-Dole B coefficient, etc.) to yield near aqueous levels of activity. Although, little is understood about the mechanistic details of salt-activation, increased enzyme flexibility and active site polarity have been hypothesized. High concentrations of salt in the pre-lyophilized enzyme solution are thought to induce preferential hydration of the enzyme and promote the retention of aqueous-like micro-pools during the freeze-drying process, thereby helping to preserve enzyme flexibility and increasing catalytic activity.;This study has investigated and characterized the aqueous-like micro pools using 2H NMR relaxation methods. The molecular dynamics of the enzyme-bound water was measured for a series of enzyme-salt formulations in different organic solvents, and it was discovered that the timescale of motion for residual water molecules on the biocatalyst &parl0;tc&parr0;D20 , in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% (w/w) CsF) as (kcat/KM) of the biocatalyst formulation increased from 0.092 s-1M-1 (salt-free) to 1140 s-1M-1 (98% (w/w) CsF). Similar effects were observed in acetone, where the timescale decreased from 24 ns (salt-free) to 2.87 ns (98% (w/w) KF) with a corresponding increase in (k cat/KM) of 0.140 s-1M-1 (salt-free) to 12.8 s-1M-1 (98% (w/w) KF). These data provide indirect evidence that salt activation is mediated by increased water mobility, as well as showing for the first time that the presence of salt alters the local mobility of the enzyme in a manner similar to that of increasing temperature, thereby affording a greater degree of transition-state flexibility for the salt-activated biocatalyst over the salt-free biocatalyst.;The present work examines protein flexibility at different timescales in insoluble solid biocatalysts and compares these results directly to biocatalyst turnover numbers and catalytic efficiencies. In order to illuminate the timescale of dynamics important to catalysis, as well as to distinguish active site polarity versus dynamical activation effects, multi-nuclear NMR spectroscopy was used to measure enzyme motions within the salt-activated biocatalyst and probe polarity effects at the active site. Three time regimes were accessed using 1H NMR relaxometry: picosecond to tens of nanoseconds (via spin lattice relaxation, T1H), microsecond to millisecond (via spin lattice relaxation in the rotating frame, (T1rhoH ), and centisecond to second (via longitudinal magnetization exchange, T1zzH). 1H NMR relaxation measurements reveal that the enzyme's turnover number (kcat) is 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 pico-nano second timescale. In addition, 19F chemical shift measurements of biocatalyst formulations inhibited with 4-fluorobenene sulfonyl fluoride suggest that enzyme activation is weakly affected by changes in active site polarity.;Independent spectroscopy experiments utilizing ESR support the 1H NMR relaxometry data. Simulations of ESR lineshapes for several salt-activated biocatalyst samples reveal two populations of enzyme in which the increases in the more dynamic population correlated with increases in biocatalyst activity. The ESR results indicate that protein dynamics measured, via a covalently bound nitroxide spin label (ESR accessible timescale is &tgr; R = 10-7 - 10-11 s), do not significantly vary from the marginally active salt-free biocatalyst (&tgr;Rfast = 0.06 ns, &tgr;Rslow = 4.2 ns, kcat/Km = 0.092 s-1M-1) to a highly active 98% (w/w) KCI biocatalyst (&tgr;Rfast = 0.06 ns, &tgr;Rslow = 3.3 ns, kcat/KM = 200.1 s-1 M-1) biocatalyst, in hexane. Active site polarity measurements of the biocatalyst samples made through measurement of the hyperfine splitting parameter, A 0, mirror similar experiments employing the 19F NMR shift to probe active site polarity, and find that enzyme activation is weakly correlated to changes in active site polarity.;Solid-state NMR was used to examine the excipent salts of biocatalysts lyophilized in the presence of variable weight percent sodium fluoride, 98% (w/w) NaC1, 98% (w/w) KF in order to detect, if possible, a population of sodium and/or fluorine ions interacting with the enzyme subtilisin Carlsberg (SC). Simple spectroscopic and NMR relaxation experiments failed shed light on the nature of these complex interactions.