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

Abstract

Deuterium spin relaxation was used to examine the motion of enzyme-bound water on subtilisin Carlsberg colyophilized 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 D2O rather than hydrogen-deuteron exchange on the protein. The results indicate that the timescale of motion for residual water molecules on the biocatalyst, ( c)D 2 O, in hexane decreased from 65 ns (salt-free) to 0.58 ns (98% CsF) as (kcat͞KM)app of the biocatalyst preparation increased from 0.092 s ؊1 ⅐M ؊1 (saltfree) to 1,140 s ؊1 ⅐M ؊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 (kcat͞KM)app of 0.140 s ؊1 ⅐M ؊1 (salt-free) to 12.8 s ؊1 ⅐M ؊1 (98% KF). Although a global correlation between water mobility and enzyme activity was not evident, linear correlations between ln[(kcat͞KM)app] and ( c)D 2 O were obtained for salt-activated enzyme preparations in both hexane and acetone. Furthermore, a direct correlation was evident between (kcat͞KM)app and the total amount of mobile water per mass of enzyme. These results suggest that increases in enzymebound 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.enzyme activation ͉ organic solvents ͉ salts ͉ subtilisin Carlsberg I n recent years, the application of selective, nonhazardous biocatalysts for chemical synthesis has become an increasingly attractive alternative to traditional chemical methods. This trend is driven in part by the exquisite chemo-, regio-, and enantioselectivities commonly demonstrated by enzymes. The high demand for enantiomerically pure and selectively functionalized molecules, especially within the pharmaceutical industry, continues to spur the expanding interest in biocatalysis. Unfortunately, many compounds of interest to the pharmaceutical and related industries exhibit poor solubility and undergo deleterious side reactions (e.g., hydrolysis) in water; hence, they are not amenable to enzymatic reactions in conventional media.Nonaqueous biocatalysis, including enzymatic reactions in nearly anhydrous organic solvents, has emerged as an alternative approach to circumvent the limitations of aqueous-based reaction systems. There are drawbacks, however, to performing enzymatic reactions in organic solvents, most notably low biocatalytic activity (1-5). Much effort has been directed toward elucidating the mechanism(s) underlying the low activity exhibited by insoluble enzyme formulations in organic solvents. Poor com...

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

“…It is noteworthy that numerous previous studies, including a recent study by our laboratory (33), provide evidence of correlations between fast solvent dynamics and molecular events evident on slower timescales [e.g., substrate dissociation in myoglobin (34); protein folding of cytochrome c, protein L, and human serum albumin (35,36); and enzyme activity (37,38)]. The intimate collusion between fast solvent dynamics and slow protein motion is referred to as solvent slaving and has been reported for many classes of proteins (39,40).…”

“…Further study of this water population indicated that the amount and motion of the residual water molecules associated with the biocatalyst correlated strongly with biocatalyst activity in an Arrhenius-like fashion, with residual water molecules in salt-activated biocatalysts exhibiting shorter correlation times for motion in the more active salt preparations (33). These water molecules exhibited correlation times in the picosecond-to-nanosecond time regime.…”

IMP Dehydrogenase: Structure, Mechanism, and Inhibition

“…K + does not change the affinity of IMP. Interestingly, water and salt are believed to act as molecular lubricants to increase enzyme activity in organic solvents 148. Perhaps K + activates IMPDH by similarly facilitating the interchange of conformations.…”

Influencing factors and partitioning of respiration in a Leymus chinensis steppe in Xilin River Basin, Inner Mongolia, China

“…The mechanism explaining the rapid release of CO 2 to the atmosphere is not well understood (Halverson et al, 2000). The potential explanations include the release of intracellular solutes to the environment by soil bacteria due to the disruption of cell membranes or cell lysis under the dramatic difference in water potential after rain (Halverson et al, 2000), the physical displacement of percolating water with CO 2 built up during the previous dry period (Huxman et al, 2004), the rapid catabolism of low molecular weight carbohydrates or other labile carbon solutes to CO 2 , the rapid increase of microbial activity (Fierer and Schimel, 2002;Saetre and Stark, 2005;Xu et al, 2004), and the increased effectiveness of enzymes and consequent reduction in Ea (active energy) (Daniel et al, 2003;Eppler et al, 2006). In a system that has not experienced rainfall for some time, where physiological activity is very low, rainfall first triggers a burst of positive CO 2 flux, caused by the combination of mechanisms discussed above.…”