title: Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme.
abstract: The leader proteinase (L(pro)) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp(163), which orients the catalytic histidine residue, and Asp(164); the equivalent residues in papain are Asn(175) and Ser(176). The importance of these residues for L(pro) activity was examined by site-directed mutagenesis. Replacement of Asp(163) with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of L(pro), eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp(164) by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8 min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp(164), and to a lesser extent that of Asp(163), is extremely important for L(pro) to attain full activity upon synthesis.
authors.affiliation: Institute of Medical Biochemistry, Division of Biochemistry, University of Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Schlick
authors.forename: Jakub
authors.initials: J
authors.lastname: Kronovetr
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hampoelz
authors.forename: Tim
authors.initials: T
authors.lastname: Skern
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry*; D004926:Escherichia coli; D039603:Eukaryotic Initiation Factor-4G; D029544:Foot-and-Mouth Disease Virus / Q000201:enzymology*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D055672:Static Electricity
chemical_list: D039603:Eukaryotic Initiation Factor-4G; D010448:Peptide Initiation Factors; D001224:Aspartic Acid; D012694:Serine; D001216:Asparagine; D010450:Endopeptidases; D010206:Papain; C088747:leader proteinase, foot-and-mouth disease virus