Rules for the regulation of enzyme activity in reserved micelles as illustrated by the conversion of apolar steroids by 20β-hydroxysteroid dehydrogenase

Authors


Abstract

20β-Hydroxysteroid dehydrogenase was enclosed in reversed micellar media consisting of cetyltrimethyl-ammonium bromide, hexanol, organic solvent and Hepes buffer. The influence of the composition of these media on the enzymatic reduction of the apolar steroids progesterone and prednisone was investigated by varying the water content, concentration of hexanol and type of organic solvent. By changing the water content and the type of organic solvent, the hexanol to cetyltrimethylammonium bromide ratio in the interphase can be varied. This ratio was determined by phase boundary titrations. It was found that the higher this ratio, the higher the rate of steroid conversion. From variations of the hexanol content it was concluded that the rate of steroid conversion is determined by the hydrophobicity of the steroid relative to the hydrophobicity of the continuous phase and the hydrophobicity of the interphase. The hydrophobicity of the phases was expressed in log P-values. Log P is defined as the logarithm of the partition coefficient in an octanol-water two-phase system. This enabled us to derive the following relations between the hydrophobicity values for the substrate (log Ps), for the interphase (log Pi) and for the continuous phase (log Pcph): |log Pi-log Ps| must be minimal to ensure a high steroid concentration in the interphase and |log Pcph-log Ps| must be large to keep the steroid concentration in the continuous phase low. With these considerations, for any given apolar compound, a medium can be composed that gives optimal enzymatic conversion.

Abbreviations and Symbols
CTAB

cetyltrimethylammonium bromide

HSDH

20β-hydroxysteroid dehydrogenase;a0, molar ratio of hexanol to CTAB in the interphase

w0

molar ratio of water to CTAB

log P

logarithm of partition coefficient in an octanol-water two-phase system

log Pi

log P value for the interface

log Pcph

log P value for the continuous phase

log Ps

log P value for the steroid

V′m

rate of reaction at infinite steroid concentration and non-saturating NADH concentration

Enzyme
 

20β-hydroxysteroid dehydrogenase (EC 1.1.1.53)

Ancillary