title: Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.
abstract: Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P(7). Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P(1)-P(3) in an extended conformation, whereas residues P(4)-P(7) are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P(5). Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes.
authors: Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Rua Tres de Maio 100, 04044-20 Sao Paulo, Brazil.
authors: Jorge A N
authors: JA
authors: Santos
authors: Iuri E
authors: IE
authors: Gouvea
authors: Wagner A S
authors: WA
authors: Júdice
authors: Mario A
authors: MA
authors: Izidoro
authors: Fabiana M
authors: FM
authors: Alves
authors: Robson L
authors: RL
authors: Melo
authors: Maria A
authors: MA
authors: Juliano
authors: Tim
authors: T
authors: Skern
authors: Luiz
authors: L
authors: Juliano
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D029544:Foot-and-Mouth Disease Virus / Q000201:enzymology*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010206:Papain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D010206:Papain; C088747:leader proteinase, foot-and-mouth disease virus