Kosmotropic salt activation and substrate specificity of poliovirus protease 3C

Kosmotropic salt activation and substrate specificity of poliovirus protease 3C

Author Gouvea, Iuri E. Google Scholar
Judice, Wagner A. S. Google Scholar
Cezari, Maria H. S. Google Scholar
Juliano, Maria A. Google Scholar
Juhasz, Tunde Google Scholar
Szeltner, Zoltan Google Scholar
Polgar, Laszlo Google Scholar
Juliano, Luiz Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Hungarian Acad Sci
Abstract Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. the peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO42- > HPO42- > acetate(-) > HCO3- > Cl-. the activation appeared to be independent of substrate sequence and arose primarily from an increase in k(cat). A shift to higher pH was also observed for the pK(1) of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.
Language English
Date 2006-10-03
Published in Biochemistry. Washington: Amer Chemical Soc, v. 45, n. 39, p. 12083-12089, 2006.
ISSN 0006-2960 (Sherpa/Romeo, impact factor)
Publisher Amer Chemical Soc
Extent 12083-12089
Origin http://dx.doi.org/10.1021/bi060793n
Access rights Closed access
Type Article
Web of Science ID WOS:000240752400037
URI http://repositorio.unifesp.br/handle/11600/29204

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