Activity and conformation changes of Chinese hamster dihydrofolate reductase in reverse micelles.
Changes in the activity and conformation of Chinese hamster dihydrofolate reductase (ch-DHFR) were studied in reverse micelles of cetyltrimethylammonium bromide (CTAB) and dodecylammonium butyrate (DAB) at various water contents and denaturant concentrations. The ch-DHFR entrapped in CTAB and DAB reverse micelles shows very low activity at a lower ratio of water to surfactant (omega(0)). The activity was enhanced in the presence of low concentrations of guanidine hydrochloride. Emission fluorescence spectra of ch-DHFR in aqueous medium and in reverse micelles in the presence of low concentrations of denaturants were compared. Only a slight change in emission intensity of ch-DHFR accompanies enzyme activation in the presence of low concentrations of guanidine hydrochloride in CTAB and DAB reverse micelles. There was no detectable change in the average diameters of CTAB and DAB reverse micelles in the presence of low concentrations of denaturants measured by laser light scattering, indicating that the enzyme activation in the presence of denaturant is not due to a size change in the reverse micelles. Our results suggest that the reduced activity of ch-DHFR in reverse micelles at low omega(0) is due to the restrictions in enzyme conformation caused by an environment with low water content. The reduction in enzyme activity was restored by the presence of low concentrations of denaturant or increased water content, indicating that conformation flexibility is important for full expression of enzyme activity.
Department of Technical Physics, Peking University, Beijing, 100871, China.
H
H
Zhu
Y X
YX
Fan
N
N
Shi
J M
JM
Zhou
D000588:Amines; D000818:Animals; D016466:CHO Cells; D000077286:Cetrimonium; D002593:Cetrimonium Compounds; D006224:Cricetinae; D003412:Cricetulus; D019791:Guanidine / Q000494:pharmacology; D066298:In Vitro Techniques; D008823:Micelles; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*
D000588:Amines; D002593:Cetrimonium Compounds; D008823:Micelles; D013762:Tetrahydrofolate Dehydrogenase; D019791:Guanidine; C003935:dodecylamine; D000077286:Cetrimonium