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Structure and activity of trypsin in reverse micelles
P Walde1, Q Peng, N W Fadnavis
1Institut für Polymere, Eidgenössische Technische Hochschule, Zürich, Switzerland.
European Journal of Biochemistry
|April 15, 1988
Summary
Investigating trypsin in reverse micelles reveals altered kinetic parameters (kcat and Km) influenced by water content and surfactant type. Enzyme activity is optimal with limited bound water, suggesting potential denaturation in micellar environments.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Surfactant chemistry
Background:
- Trypsin's kinetic properties are crucial for understanding its catalytic mechanisms.
- Reverse micelles offer a unique microenvironment for studying enzyme behavior.
- Previous studies on alpha-chymotrypsin showed enhanced activity in similar systems.
Purpose of the Study:
- To investigate the kinetic properties of trypsin in two different reverse micellar systems.
- To compare trypsin's behavior in reverse micelles with its activity in aqueous solutions and with alpha-chymotrypsin.
- To elucidate the impact of micellar conditions, particularly water content, on trypsin's catalytic efficiency and structure.
Main Methods:
- Enzyme kinetics studies using three different substrates (N alpha-benzoyl-L-Arg ethyl ester, BzPheValArg-NH-Np, ZLysO-Np).
- Utilized bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and hexadecyltrimethyl ammonium bromide (CTAB) as surfactants in specific solvent systems.
- Employed rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies.
- Spectroscopic analyses including ultraviolet absorption, circular dichroism (CD), and fluorescence as a function of water content (wo).
Main Results:
- Kinetic parameters (kcat and Km) varied significantly and were affected by micellar conditions and water content (wo).
- Substrate BzPheValArg-NH-Np showed a much smaller kcat in reverse micelles compared to water.
- Substrate ZLysO-Np exhibited a slight enhancement in kcat at pH 3.2 in CTAB micelles, attributed to a shift in the rate-limiting step.
- Maximal enzyme activity was observed at low wo values (below 12), indicating a preference for limited hydration.
- Spectroscopic studies suggested partial unfolding of trypsin in AOT micelles, potentially due to calcium ion depletion, with further unfolding at higher wo values.
Conclusions:
- The micellar environment significantly alters trypsin's kinetic parameters and can induce structural changes.
- Optimal trypsin activity in reverse micelles occurs at low water content, suggesting a specific hydration shell is beneficial.
- The observed changes in kinetic and structural properties highlight the influence of the microenvironment on enzyme function.
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