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

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 ⌬⌬S † , for the more active biocatalyst preparations in organic solvents. In contrast, ⌬⌬H † 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.enzyme activation ͉ enzyme dynamics ͉ NMR spectroscopy ͉ organic solvents ͉ subtilisin Carlsberg T he direct coupling of protein dynamics to enzymatic catalysis is strongly supported by recent studies of enzymes in aqueous solution (1-3). These studies have revealed insights into the connection between enzyme activity and protein dynamics [e.g., conformation exchange in CypA (4), Aquifex Adk lid movement (5), Met-20 loop dynamics in DHFR (6), loop motions leading to conformational change in RNase A (7), and modulation of surfaceglycosylated chymotrypsin structural dynamics and activity (8)], yet the relatively small range of enzyme activities accessible under aqueous conditions, without the use of protein modifications or alteration of the solvent, hinders thorough investigation of the central hypothesis that enzyme catalysis is intimately coupled to protein dynamics. One strategy for broadening the range of activities over which enzyme catalysis and protein dynamics can be studied concurrently is to employ nonaqueous or organic solvents. For example, a correlation between molecular dynamics, as evidenced by a reduced rate of amide H/D exchange, and biocatalyst activity has been observed for PEGylated subtilisin Carlsberg (SC) in 1,4-dioxane (9).Nonaqueous biocata...

Nature Versus Nurture: Developing Enzymes That Function Under Extreme Conditions

“…Protein motions play an important role in myriad protein functions, including catalysis and signaling (23); therefore, altered enzyme dynamics in organic solvents can have a detrimental effect on biocatalytic activity. Restricted motion has been strongly correlated with enzyme activity, or lack thereof, in organic solvents (24)(25)(26). Structural data available for proteins in organic solvents are limited when compared with the vast array of complete structures available for proteins in water.…”

“…Overall or local changes in the secondary or tertiary structure of proteins in organic solvents are typically detected using Fourier transform infrared spectroscopy (27), circular dichroism (CD) (28), fluorescence, and electron spin resonance spectroscopy (29,30), but there are few reports of data collected at the atomic level. NMR has the ability to provide higher-resolution information but to date has provided only bulk-average data (25), surface water properties (31), or single-site information (26). In one published high-resolution study, a crystal structure of the protease subtilisin Carlsberg formed in water and soaked in acetonitrile showed almost no structural change from the aqueous structure (32).…”

Enzyme activity in liquid lipase melts as a step towards solvent-free biology at 150 °C

“…Conversely, in non-aqueous media, lipases can use other nucleophiles, such as methanol, to catalyse esterification and transesterification reactions [2][3][4][5][6][7][8][9][10][11][12][13] , although the explicit removal of water is not often a priority. Typically, enzyme reactions performed in organic solvents still contain a hydration layer at the surface of the protein, and the pervasiveness of the solvent in these examples highlights the requirement for a substrate delivery medium [14][15][16][17][18][19][20][21][22][23][24][25] . There have also been reports of lipase catalysis where the reaction proceeds with solvent quantities of one of the reagents 3,12,13 , where the lipases are typically present as either a lyophilized powder 12,13,26 , or immobilized on a substrate 2,3,8,10,27,28 , but there are no examples of molecularly dispersed enzymes catalysing reactions in the complete absence of a solvent.…”

Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg

“…The implications of theses findings is that while the enzyme becomes more rigid by the removal of its internal water molecules over a 96 hour period, the active site polarity should decrease (as suggested by our fluorescence data) and disruption of the oxyanion hole might reduce the enzyme’s ability to stabilize the tetrahedral intermediates, decreasing both V max and K M , as shown by the results here presented. Furthermore, a decrease in the polarity of the active site and disruption of the oxyanion hole could lead to reorientation of the bound substrate [12] or allow the substrate to bind in a less restricted fashion, letting it move more freely, as it has been shown on previous studies involving EPR spectroscopy [9, 31]. In short, one interpretation that is emerging from these studies is that changes in active site polarity during prolonged exposure to organic solvents are the major cause of the observed decrease in enzymatic activity.…”

“…Nevertheless, it is important to discuss some of these results since the effects that are linked to enzyme activity are enzyme flexibility and active-site polarity, both related to our work. Eppler et al 19 F NMR active site polarity studies show that increasing the salt concentration (NaCl, NaF, KCl and KF) in hexane from 0 to 98% did not augment enzyme polarity (while enzyme activity was increased), concluding that changes in polarity do not affect enzyme activity [31]. On a similar and more recent study however, it was concluded that active site polarity and hydration do affect enzyme activity, but that this effect was also dependent on the solvent used [32].…”