title: Increase of SARS-CoV 3CL peptidase activity due to macromolecular crowding effects in the milieu composition.
abstract: The 3C-like peptidase of the severe acute respiratory syndrome virus (SARS-CoV) is strictly required for viral replication, thus being a potential target for the development of antiviral agents. In contrast to monomeric picornavirus 3C peptidases, SARS-CoV 3CLpro exists in equilibrium between the monomer and dimer forms in solution, and only the dimer is proteolytically active in dilute buffer solutions. In this study, the increase of SARS-CoV 3CLpro peptidase activity in presence of kosmotropic salts and crowding agents is described. The activation followed the Hofmeister series of anions, with two orders of magnitude enhancement in the presence of Na₂SO₄, whereas the crowding agents polyethylene glycol and bovine serum albumin increased the hydrolytic rate up to 3 times. Kinetic determinations of the monomer dimer dissociation constant (K(d)) indicated that activation was a result of a more active dimer, without significant changes in K(d) values. The activation was found to be independent of substrate length and was derived from both k(cat) increase and K(m) decrease. The viral peptidase activation described here could be related to the crowded intracellular environment and indicates a further fine-tuning mechanism for biological control, particularly in the microenvironment of the vesicles that are induced in host cells during positive strand RNA virus infection.
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, Brazil.
authors.forename: Debora N
authors.initials: DN
authors.lastname: Okamoto
authors.forename: Lilian C G
authors.initials: LC
authors.lastname: Oliveira
authors.forename: Marcia Y
authors.initials: MY
authors.lastname: Kondo
authors.forename: Maria H S
authors.initials: MH
authors.lastname: Cezari
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Szeltner
authors.forename: Tünde
authors.initials: T
authors.lastname: Juhász
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: László
authors.initials: L
authors.lastname: Polgár
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Iuri E
authors.initials: IE
authors.lastname: Gouvea
mesh_terms: D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000201:enzymology* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases