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Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach
Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we hav...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924875/ https://www.ncbi.nlm.nih.gov/pubmed/27352302 http://dx.doi.org/10.1371/journal.pone.0158024 |
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author | Dutta, Sruti Choudhury, Debi Roy, Sumana Dattagupta, Jiban Kanti Biswas, Sampa |
author_facet | Dutta, Sruti Choudhury, Debi Roy, Sumana Dattagupta, Jiban Kanti Biswas, Sampa |
author_sort | Dutta, Sruti |
collection | PubMed |
description | Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity—I86F and I86L have 2.7 and 29.1 times higher k(cat)/K(m) values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. |
format | Online Article Text |
id | pubmed-4924875 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-49248752016-07-18 Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach Dutta, Sruti Choudhury, Debi Roy, Sumana Dattagupta, Jiban Kanti Biswas, Sampa PLoS One Research Article Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity—I86F and I86L have 2.7 and 29.1 times higher k(cat)/K(m) values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. Public Library of Science 2016-06-28 /pmc/articles/PMC4924875/ /pubmed/27352302 http://dx.doi.org/10.1371/journal.pone.0158024 Text en © 2016 Dutta et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Dutta, Sruti Choudhury, Debi Roy, Sumana Dattagupta, Jiban Kanti Biswas, Sampa Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach |
title | Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach |
title_full | Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach |
title_fullStr | Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach |
title_full_unstemmed | Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach |
title_short | Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity—A Structural and Biochemical Approach |
title_sort | mutation in the pro-peptide region of a cysteine protease leads to altered activity and specificity—a structural and biochemical approach |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924875/ https://www.ncbi.nlm.nih.gov/pubmed/27352302 http://dx.doi.org/10.1371/journal.pone.0158024 |
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