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On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.

Castillo B, Bansal V, Ganesan A, Halling P, Secundo F, Ferrer A, Griebenow K, Barletta G - BMC Biotechnol. (2006)

Bottom Line: Additionally, the hydration level of the enzyme does not seem to affect its stability.Incubation was also accompanied by a substantial decrease in Vmax/KM.These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes.

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Affiliation: Department of Chemistry, University of Puerto Rico at Humacao, CUH Station, Humacao, 00791, Puerto Rico. betzaida.castillo@yahoo.com <betzaida.castillo@yahoo.com>

ABSTRACT

Background: Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.

Results: As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-beta-cyclodextrin (MbetaCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.

Conclusion: These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

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Structure of 4-Ethoxyfluorophosphinyloxy-TEMPO.
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Figure 8: Structure of 4-Ethoxyfluorophosphinyloxy-TEMPO.

Mentions: For slow molecular motions (τ > 3 × 10-9 s), the line positions depend on the rate as well as the amplitude of motion. Hence, the parameter ΔH0 can be used as a convenient empirical measure of dynamics, including both amplitude and rate of motion (a decrease in ΔH0 indicates increased degree of freedom). In addition, the parameters hi and ha (spectra shown on (Figure 7) can also be used to determine the freedom of rotation of the spin label (a high value of hi/(hi+ha) indicates higher degree of freedom). For our studies, lyophilized subtilisin Carlsberg was spin-labeled at the active site with a classic inhibitor for serine proteases: 4-ethoxyfluorophosphinyloxy-TEMPO (Figure 8). After labeling, activity and active site titrations were performed to ensure the enzyme was in fact labeled at the active-site (Materials and Methods). Our results, summarized on (Figure 7), indicate that the degree of freedom (or mobility) of the spin label, bound to the enzyme's active site, increases during the five days of incubation. Although Tau could not be measured at day 0 (no incubation), ΔH0 and hi/(hi+ha) show that the mobility of the spin label increases after a 4-day incubation, suggesting increased flexibility of the enzyme or "over relaxation" of the active site. Besides the activity loss observed, increased mobility of the spin label seems to be the only consequence of incubation in 1,4-dioxane, and it could therefore be interpreted as the cause for the low enzyme stability. If, however, one argues that increased mobility of the spin-label relates to increased enzyme flexibility, then these results seem to be contradictory to the general belief that increasing the flexibility should increase the enzyme's activity, unless the enzyme's flexibility was already at its optimum before incubation, and increased flexibility would only cause a decrease in activity. But perhaps the most plausible explanation is that the enzyme's active-site is becoming distorted or "over relaxed" during incubation, and this results in a decrease in Vmax/KM, in initial activity and an increase in apparent flexibility of the spin label is observed. Since added water has been reported to increase an enzyme's flexibility [25,26], changes of hydration water from the enzyme (occurring during the incubation period) could explain the increased flexibility observed after the 5-day incubation period. To determine this, we conducted a series of incubation and EPR measurements in 1,4-dioxane + 0.5% of added water and controlling its water activity using hydrated BaBr2 salts.


On the activity loss of hydrolases in organic solvents: II. a mechanistic study of subtilisin Carlsberg.

Castillo B, Bansal V, Ganesan A, Halling P, Secundo F, Ferrer A, Griebenow K, Barletta G - BMC Biotechnol. (2006)

Structure of 4-Ethoxyfluorophosphinyloxy-TEMPO.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764882&req=5

Figure 8: Structure of 4-Ethoxyfluorophosphinyloxy-TEMPO.
Mentions: For slow molecular motions (τ > 3 × 10-9 s), the line positions depend on the rate as well as the amplitude of motion. Hence, the parameter ΔH0 can be used as a convenient empirical measure of dynamics, including both amplitude and rate of motion (a decrease in ΔH0 indicates increased degree of freedom). In addition, the parameters hi and ha (spectra shown on (Figure 7) can also be used to determine the freedom of rotation of the spin label (a high value of hi/(hi+ha) indicates higher degree of freedom). For our studies, lyophilized subtilisin Carlsberg was spin-labeled at the active site with a classic inhibitor for serine proteases: 4-ethoxyfluorophosphinyloxy-TEMPO (Figure 8). After labeling, activity and active site titrations were performed to ensure the enzyme was in fact labeled at the active-site (Materials and Methods). Our results, summarized on (Figure 7), indicate that the degree of freedom (or mobility) of the spin label, bound to the enzyme's active site, increases during the five days of incubation. Although Tau could not be measured at day 0 (no incubation), ΔH0 and hi/(hi+ha) show that the mobility of the spin label increases after a 4-day incubation, suggesting increased flexibility of the enzyme or "over relaxation" of the active site. Besides the activity loss observed, increased mobility of the spin label seems to be the only consequence of incubation in 1,4-dioxane, and it could therefore be interpreted as the cause for the low enzyme stability. If, however, one argues that increased mobility of the spin-label relates to increased enzyme flexibility, then these results seem to be contradictory to the general belief that increasing the flexibility should increase the enzyme's activity, unless the enzyme's flexibility was already at its optimum before incubation, and increased flexibility would only cause a decrease in activity. But perhaps the most plausible explanation is that the enzyme's active-site is becoming distorted or "over relaxed" during incubation, and this results in a decrease in Vmax/KM, in initial activity and an increase in apparent flexibility of the spin label is observed. Since added water has been reported to increase an enzyme's flexibility [25,26], changes of hydration water from the enzyme (occurring during the incubation period) could explain the increased flexibility observed after the 5-day incubation period. To determine this, we conducted a series of incubation and EPR measurements in 1,4-dioxane + 0.5% of added water and controlling its water activity using hydrated BaBr2 salts.

Bottom Line: Additionally, the hydration level of the enzyme does not seem to affect its stability.Incubation was also accompanied by a substantial decrease in Vmax/KM.These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Puerto Rico at Humacao, CUH Station, Humacao, 00791, Puerto Rico. betzaida.castillo@yahoo.com <betzaida.castillo@yahoo.com>

ABSTRACT

Background: Enzymes have been extensively used in organic solvents to catalyze a variety of transformations of biological and industrial significance. It has been generally accepted that in dry aprotic organic solvents, enzymes are kinetically trapped in their conformation due to the high-energy barrier needed for them to unfold, suggesting that in such media they should remain catalytically active for long periods. However, recent studies on a variety of enzymes demonstrate that their initial high activity is severely reduced after exposure to organic solvents for several hours. It was speculated that this could be due to structural perturbations, changes of the enzyme's pH memory, enzyme aggregation, or dehydration due to water removal by the solvents. Herein, we systematically study the possible causes for this undesirable activity loss in 1,4-dioxane.

Results: As model enzyme, we employed the protease subtilisin Carlsberg, prepared by lyophilization and colyophilization with the additive methyl-beta-cyclodextrin (MbetaCD). Our results exclude a mechanism involving a change in ionization state of the enzyme, since the enzyme activity shows a similar pH dependence before and after incubation for 5 days in 1,4-dioxane. No apparent secondary or tertiary structural perturbations resulting from prolonged exposure in this solvent were detected. Furthermore, active site titration revealed that the number of active sites remained constant during incubation. Additionally, the hydration level of the enzyme does not seem to affect its stability. Electron paramagnetic resonance spectroscopy studies revealed no substantial increase in the rotational freedom of a paramagnetic nitroxide inhibitor bound to the active site (a spin-label) during incubation in neat 1,4-dioxane, when the water activity was kept constant using BaBr2 hydrated salts. Incubation was also accompanied by a substantial decrease in Vmax/KM.

Conclusion: These results exclude some of the most obvious causes for the observed low enzyme storage stability in 1,4-dioxane, mainly structural, dynamics and ionization state changes. The most likely explanation is possible rearrangement of water molecules within the enzyme that could affect its dielectric environment. However, other mechanisms, such as small distortions around the active site or rearrangement of counter ions, cannot be excluded at this time.

Show MeSH
Related in: MedlinePlus