The leader proteinase (Lpro) 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 Asp163, which orients the catalytic histidine residue, and Asp164; the equivalent residues in papain are Asn175 and Ser176. The importance of these residues for Lpro activity was examined by site-directed mutagenesis. Replacement of Asp163 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 Lpro, eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp164 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 8min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp164, and to a lesser extent that of Asp163, is extremely important for Lpro to attain full activity upon synthesis.
- enzyme mechanism
- cysteine proteinase
- RNA virus
- translation initiation
↵1 Present address: Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.
Abbreviations used: AMPSO, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulphonic acid; Caps, 3-(cyclohexylamino)propane-1-sulphonic acid; AMC, 7-amino-4-methylcoumarin; eIF, eukaryotic initiation factor; FMDV, foot-and-mouth-disease virus; IRES, internal ribosomal entry site; Lpro, leader proteinase; Pap-Cys (etc.), papain residue Cys (etc.); RRL, rabbit reticulocyte lysate; D163N (etc.), Asp163→asparagine mutant (etc.).
- The Biochemical Society, London ©2002