The role of conformational flexibility of enzymes in the discrimination between amino acid and ester substrates for the subtilisin-catalyzed reaction in organic solvents

Watanabe, Keita; Yoshida, Takashi; Ueji, Shinichi

doi:10.1016/j.bioorg.2004.05.001

Cited by 30 publications

(16 citation statements)

References 40 publications

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“…However, a more complex two or more component spectrum is generally observed when the spin label is in the active site, and the nitroxide side chain interacts with the active-site residues side groups. The results here presented show the classical two component spectra previously observed with similar spin labels and enzymes [14,16,17,18,19]: A rigid and a mobile component. The rigid component diminishes considerably while the mobile one increases when the two enzyme preparations here used (the lyophilized powder and the PEG 5000 chemically modified enzyme) are stored in the solvents for 96 h (except for the PEG-enzyme in ACN were the mobile component predominates during the storage experiments).…”

Section: Introductionsupporting

confidence: 84%

“…The study was completed with the lyophilized powder (for which much kinetic and structural data is available) and with the more active and enantioselective PEG 5000 -chemically modified enzyme. EPR spectroscopy is frequently employed to learn about the dynamics of enzymes suspended in non-aqueous media, and in most cases such information is extracted from a characteristic two component spectra similar to ours [18,19,26]. The components are usually explained as corresponding to spin labels having different freedoms of rotation within the same sample (isotropic and anisotropic motions) [27].…”

Section: Resultsmentioning

confidence: 99%

“…It has been postulated that a slow moving spin label (rigid) generates the anisotropic component, while a freely moving one (or mobile) gives rice to the isotropic part of the spectrum. The ratio of the anisotropic and isotropic pecks (Figure 1, PEG-enzyme in THF) have been used as a measure of the enzyme flexibility by the relationship Hi/(Hi+Ha) [19]. This relationship has been used throughout our studies to determine the freedom of rotation of the spin label bound to subtilisin Carlsberg active site as a function of its exposure to organic solvents.…”

Section: Methodsmentioning

confidence: 99%

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Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

et al. 2012

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The potential of enzyme catalysis in organic solvents for synthetic applications has been overshadowed by the fact that their catalytic properties are affected by organic solvents. In addition, it has recently been shown that an enzyme’s initial activity diminishes considerably after prolonged exposure to organic media. Studies geared towards understanding this last drawback have yielded unclear results. In the present work we decided to use electron paramagnetic resonance spectroscopy (EPR) to study the motion of an active site spin label (a nitroxide free radical) during 96 h of exposure of the serine protease subtilisin Carlsberg to four different organic solvents. Our EPR data shows a typical two component spectra that was quantified by the ratio of the anisotropic and isotropic signals. The isotropic component, associated with a mobile nitroxide free radical, increases during prolonged exposure to all solvents used in the study. The maximum increase (of 43%) was observed in 1,4-dioxane. Based on these and previous studies we suggest that prolonged exposure of the enzyme to these solvents provokes a cascade of events that could induce substrates to adopt different binding conformations. This is the first EPR study of the motion of an active-site spin label during prolonged exposure of an enzyme to organic solvents ever reported.

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Section: Introductionsupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

et al. 2012

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“…26 Due to the rigid carrier of I-CALB, thus, exposure to a magnetic field has little influence on the secondary structure of I-CALB. Watanabe et al 27 noted that the flexibility of an enzyme may be critical for its activity. There are two water molecules around the active site of CALB, 28 which will affect flexibility of CALB.…”

Section: Resultsmentioning

confidence: 99%

Effects of magnetic fields on the enzymatic synthesis of naringin palmitate

Zhang

Wan

et al. 2018

RSC Adv.

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“…Currently, we hypothesize that surfactant aggregate at concentrations of 3.0 m m form structures which directly interact with the protein structure. This may result from interactions with solvent molecular motions or with hydrophobic partition within the in micelle core, possibly, resulting in greater flexibility throughout the enzyme structure, improving selectivity and activity . In this study we compare APG micelle structure evolution as a function of concentration and compare them with corresponding changes in protease and HRP activity.…”

Section: Introductionmentioning

confidence: 99%

Enzyme Activity and Structural Dynamics Linked to Micelle Formation: A Fluorescence Anisotropy and ESR Study

Chin

Somasundaran

2013

Photochem & Photobiology

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Activities of the enzymes, protease subtilisin and horse radish peroxidase (HRP) have been increased 50 and 40%, respectively, in the presence of the nonionic surfactant, alkyl polyglucoside, compared with the activities in buffer alone. This enzyme hyperactivity reaches a peak at 3.0 mm of surfactant. Investigation into the structure of surfactant aggregates indicates "giant" micelle superstructures at this range of surfactant concentration of 1.7 μm in diameter--dramatically decreasing to 60 and 70 nm at higher surfactant concentrations, while surface tension measurements indicate two critical micelle concentration inflection points at 0.2 and 5.0 mm, which suggests transitions in micelle structure with respect to concentration. Furthermore, electron spin resonance (ESR) indicates that the micelles in first critical micelle concentration regime are loosely packed relative to the second aggregate phase. We hypothesize that this loose packing results in diminished hydration shell repulsion between the micelles, leading to the large, micrometer-sized aggregates. We further hypothesize that it is the interaction with these loosely packed micelles that affects the flexibility of the HRP and protease enzyme structure. Time-resolved fluorescence anisotropy of subtilisin in Brij-30 indicates increasing flexibility of catalytic active site with surfactant concentration. This is correlated with an increase in enzymatic activity.

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The role of conformational flexibility of enzymes in the discrimination between amino acid and ester substrates for the subtilisin-catalyzed reaction in organic solvents

Cited by 30 publications

References 40 publications

Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

Low Operational Stability of Enzymes in Dry Organic Solvents: Changes in the Active Site Might Affect Catalysis

Effects of magnetic fields on the enzymatic synthesis of naringin palmitate

Enzyme Activity and Structural Dynamics Linked to Micelle Formation: A Fluorescence Anisotropy and ESR Study

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