INVESTIGADORES
CELEJ Maria Soledad
artículos
Título:
Superactivity and conformational changes on alpha-chymotrypsin upon interfacial binding to cationic micelles.
Autor/es:
CELEJ MS, D´ANDREA MG, CAMPANA PT, FIDELIO GD, BIANCONI ML.
Revista:
BIOCHEMICAL JOURNAL
Editorial:
Biochemical Society
Referencias:
Año: 2004 vol. 378 p. 1059 - 1066
ISSN:
0264-6021
Resumen:
<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0in; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:ES; mso-fareast-language:ES;} @page Section1 {size:8.5in 11.0in; margin:1.0in 1.25in 1.0in 1.25in; mso-header-margin:.5in; mso-footer-margin:.5in; mso-paper-source:0;} div.Section1 {page:Section1;} --> The catalytic behaviour of α-CT (α-chymotrypsin) is affected by cationic micelles of CTABr (hexadecyltrimethylammonium bromide). The enzyme–micelle interaction leads to an increase in both the Vmax and the affinity for the substrate p-nitrophenyl acetate, indicating higher catalytic efficiency for bound α-CT. The bell-shaped profile of α-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 α-CT conformation. Changes in tertiary structure were observed by the increase in intensity and the red shift in the α-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 α-helix, as seen by far-UV CD and FTIR (Fourier-transform infrared) spectroscopies. The far-UV CD spectrum of α-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 α-helical content. This is in agreement with FTIR studies, whereas an increase in the band at 1655 cm−1, corresponding to the α-helix, was shown by fitting analysis and difference spectroscopy. Spectral deconvolution indicated a reduction in the β-sheet content in micelle-bound α-CT. Our data suggest that the higher catalytic efficiency of micelle-bound α-CT results from significant conformational changes.