Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles.
The catalytic behaviour of alpha-CT (alpha-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme-micelle interaction leads to an increase in both the V(max) and the affinity for the substrate p -nitrophenyl acetate, indicating higher catalytic efficiency for bound alpha-CT. The bell-shaped profile of alpha-CT activity with increasing CTABr concentrations suggests that the micelle-bound enzyme reacts with the free substrate. Although more active with CTABr micelles, the enzyme stability is essentially the same as observed in buffer only. Enzyme activation is accompanied by changes in alpha-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the alpha-CT tryptophan fluorescence spectrum, suggesting the annulment of internal quenching and a more polar location of tryptophan residues. Near-UV CD also indicated the transfer of aromatic residues to a more flexible environment. CTABr micelles also induces an increase in alpha-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of alpha-CT shows an increase in the intensity of the positive band at 198 nm and in the negative band at 222 nm, indicating an increased alpha-helical content. This is in agreement with FTIR studies, where an increase in the band at 1655 cm(-1), corresponding to the alpha-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the beta-sheet content in micelle-bound alpha-CT. Our data suggest that the higher catalytic efficiency of micelle-bound alpha-CT results from significant conformational changes.
Celej
M Soledad
MS
Departamento de Química Biológica-CIQUIBIC, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
D'Andrea
Mariana G
MG
Campana
Patricia T
PT
Fidelio
Gerardo D
GD
Bianconi
M Lucia
ML
D002412:Cations / Q000378:metabolism; D000077286:Cetrimonium; D002593:Cetrimonium Compounds / Q000378:metabolism; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008823:Micelles; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D002412:Cations; D002593:Cetrimonium Compounds; D008823:Micelles; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D000077286:Cetrimonium