Characterization of active-site residues of the NIa protease from tobacco vein mottling virus

Duk Chul Hwang, Do Hyung Kim, Jeong Soo Lee, Byoung Heon Kang, Jiseon Han, Woong Kim, Byeung Doo Song, Kwan Yong Choi

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.

Original languageEnglish (US)
Pages (from-to)505-511
Number of pages7
JournalMolecules and cells
Volume10
Issue number5
DOIs
StatePublished - Oct 31 2000

Bibliographical note

Funding Information:
Acknowledgments This work was supported partly by BK21 Program from Ministry of Education and by the R & D Promotion Center Agricultural.

Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.

Keywords

  • NIa protease
  • Tobacco vein mottling virus

Fingerprint Dive into the research topics of 'Characterization of active-site residues of the NIa protease from tobacco vein mottling virus'. Together they form a unique fingerprint.

Cite this