Hydrolytic Properties and Substrate Specificity of the Foot-and-Mouth Disease Leader Protease

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• 作者：Jorge A. N. Santos ; Iuri E. Gouvea ; Wagner A. S. Jdice ; Mario A. Izidoro ; Fabiana M. Alves ; Robson L. Melo ; Maria A. Juliano ; Tim Skern ; Luiz Juliano
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：August 25, 2009
• 年：2009
• 卷：48
• 期：33
• 页码：7948-7958
• 全文大小：970K
• 年卷期：v.48,no.33(August 25, 2009)
• ISSN：1520-4995

文摘

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 P₇. Indeed, almost all substitutions investigated were detrimental to Lb^pro activity. Analysis of structural data showed that Lb^pro binds residues P₁−P₃ in an extended conformation, whereas residues P₄−P₇ are bound in a short 3₁₀ helix. The specificity of Lb^pro as revealed by the substituted peptides could be explained for all positions except P₅. 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