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.

The Leader Proteinase of Foot-and-Mouth Disease Virus Negatively Regulates the Type I Interferon Pathway by Acting as a Viral Deubiquitinase

“…We considered the possibility that the DUB activity of Lb pro is coupled with or dependent upon its ability to cleave eIF-4G. Previous studies revealed that mutations in amino acid residues C51, D163, and D164 in L pro partially reduced (D163N and D164N) or completely eliminated (C51A and D163N/D164N) the abilities of L pro to process itself from viral polyprotein and to cleave eIF-4G (15,41). Such activities of L pro , however, were not affected upon disruption of the SAP domain by double amino acid substitutions at residues 83 and 86 (I83A/L86A) (11).…”

Foot‐and‐mouth disease virus leader proteinase: a papain‐like enzyme requiring an acidic environment in the active site

“…2, Table 2 ). Nevertheless, the D164E mutant protein was more active than the D164N or D164A mutant proteins [12].…”

“…We have previously shown that the single mutation D163N slightly reduced self‐processing, without affecting eIF4GI cleavage. In contrast, the substitution D164N led to more drastic changes in both activities [12]. We wished to find out whether the charge and/or the geometry of residue Asp164 was important for enzyme activity.…”

“…b Cys51 was replaced by alanine in the crystal structure of Lb pro [6]. c The e¡ect of mutating residue Asp163 is described in [12]. puri¢ed the Lb pro D164E mutant and determined its speci¢city constant k cat /K m on the peptide VQRKLK-AMC to be 98 M 31 s 31 , about 25-fold lower than that of the wild-type enzyme (2.65U10 3 M 31 s 31 [12]).…”

“…These di¡erences in the position of the acidic residues have consequences for catalysis. Substitution of Asp164 with asparagine or alanine severely a¡ect L pro activity ; in contrast, replacement of the equivalent P-Ser176 with alanine only reduced papain activity two-fold when measured on a peptide substrate [12,13].…”