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Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay

FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity follo...

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Main Authors: Cahyo Budiman, Goh, Carlmond Kah Wun, Lee, Ping Chin, Rafida Razali, Thean, Chor Leow
Format: Article
Language:English
English
Published: Borneo International Journal of Biotechnology (BIJB) 2020
Subjects:
QC Physics
Online Access:https://eprints.ums.edu.my/id/eprint/27448/1/Reassessment%20of%20the%20Catalytic%20activity%20and%20substrate%20specificity%20of%20FKBP35%20from%20Plasmodium%20Knowlesi%20using%20Protease-Free%20Assay%20abstract.pdf
https://eprints.ums.edu.my/id/eprint/27448/2/Reassessment%20of%20the%20Catalytic%20activity%20and%20substrate%20specificity%20of%20FKBP35%20from%20Plasmodium%20Knowlesi%20using%20Protease-Free%20Assay%20FULLTEXT.pdf
https://eprints.ums.edu.my/id/eprint/27448/
https://jurcon.ums.edu.my/ojums/index.php/bijb/article/view/2602
https://doi.org/10.51200/bijb.vi.2602
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spelling my.ums.eprints.274482021-06-26T01:42:12Z https://eprints.ums.edu.my/id/eprint/27448/ Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay Cahyo Budiman Goh, Carlmond Kah Wun Lee, Ping Chin Rafida Razali Thean, Chor Leow QC Physics FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity followed and the tetratricopeptide repeat domain for its dimerization. The protease-coupling and protease-free assays are known to be the common methods for investigating the catalytic properties of PPIase. Earlier, the protease-coupling assay was used to confirm the catalytic activity of Pk-FKBP35 in accelerating cis-trans isomerization of the peptide substrate. This report is aimed to re-assess the catalytic and substrate specificity of Pk-FKBP35 using an alternative method of a protease-free assay. The result indicated that while Pk-FKBP35 theoretically contained many possible cleavage sites of chymotrypsin, experimentally, the catalytic domain was relatively stable from chymotrypsin. Furthermore, under protease-free assay, Pk-FKBP35 also demonstrated remarkable PPIase catalytic activity with kcat/KM of 4.5 + 0.13 × 105 M−1 s−1, while the kcat/KM of active site mutant of D55A is 0.81 + 0.05 × 105 M−1 s−1. These values were considered comparable to kcat/KM obtained from the protease-coupling assay. Interestingly, the substrate specificities of Pk-FKBP35 obtained from both methods are also similar, with the preference of Pk-FKBP35 towards Xaa at P1 position was Leu>Phe>Lys>Trp>Val>Ile>His>Asp>Ala>Gln>Glu. Altogether, we proposed that protease-free and protease-coupling assays arereliable for Pk-FKBP35. Borneo International Journal of Biotechnology (BIJB) 2020 Article PeerReviewed text en https://eprints.ums.edu.my/id/eprint/27448/1/Reassessment%20of%20the%20Catalytic%20activity%20and%20substrate%20specificity%20of%20FKBP35%20from%20Plasmodium%20Knowlesi%20using%20Protease-Free%20Assay%20abstract.pdf text en https://eprints.ums.edu.my/id/eprint/27448/2/Reassessment%20of%20the%20Catalytic%20activity%20and%20substrate%20specificity%20of%20FKBP35%20from%20Plasmodium%20Knowlesi%20using%20Protease-Free%20Assay%20FULLTEXT.pdf Cahyo Budiman and Goh, Carlmond Kah Wun and Lee, Ping Chin and Rafida Razali and Thean, Chor Leow (2020) Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay. Borneo International Journal of Biotechnology (BIJB), 1. 125 -143. ISSN 2716-697X https://jurcon.ums.edu.my/ojums/index.php/bijb/article/view/2602 https://doi.org/10.51200/bijb.vi.2602
institution Universiti Malaysia Sabah
building UMS Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Sabah
content_source UMS Institutional Repository
url_provider http://eprints.ums.edu.my/
language English
English
topic QC Physics
spellingShingle QC Physics
Cahyo Budiman
Goh, Carlmond Kah Wun
Lee, Ping Chin
Rafida Razali
Thean, Chor Leow
Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay
description FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity followed and the tetratricopeptide repeat domain for its dimerization. The protease-coupling and protease-free assays are known to be the common methods for investigating the catalytic properties of PPIase. Earlier, the protease-coupling assay was used to confirm the catalytic activity of Pk-FKBP35 in accelerating cis-trans isomerization of the peptide substrate. This report is aimed to re-assess the catalytic and substrate specificity of Pk-FKBP35 using an alternative method of a protease-free assay. The result indicated that while Pk-FKBP35 theoretically contained many possible cleavage sites of chymotrypsin, experimentally, the catalytic domain was relatively stable from chymotrypsin. Furthermore, under protease-free assay, Pk-FKBP35 also demonstrated remarkable PPIase catalytic activity with kcat/KM of 4.5 + 0.13 × 105 M−1 s−1, while the kcat/KM of active site mutant of D55A is 0.81 + 0.05 × 105 M−1 s−1. These values were considered comparable to kcat/KM obtained from the protease-coupling assay. Interestingly, the substrate specificities of Pk-FKBP35 obtained from both methods are also similar, with the preference of Pk-FKBP35 towards Xaa at P1 position was Leu>Phe>Lys>Trp>Val>Ile>His>Asp>Ala>Gln>Glu. Altogether, we proposed that protease-free and protease-coupling assays arereliable for Pk-FKBP35.
format Article
author Cahyo Budiman
Goh, Carlmond Kah Wun
Lee, Ping Chin
Rafida Razali
Thean, Chor Leow
author_facet Cahyo Budiman
Goh, Carlmond Kah Wun
Lee, Ping Chin
Rafida Razali
Thean, Chor Leow
author_sort Cahyo Budiman
title Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay
title_short Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay
title_full Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay
title_fullStr Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay
title_full_unstemmed Reassessment of the Catalytic activity and substrate specificity of FKBP35 from Plasmodium Knowlesi using Protease-Free Assay
title_sort reassessment of the catalytic activity and substrate specificity of fkbp35 from plasmodium knowlesi using protease-free assay
publisher Borneo International Journal of Biotechnology (BIJB)
publishDate 2020
url https://eprints.ums.edu.my/id/eprint/27448/1/Reassessment%20of%20the%20Catalytic%20activity%20and%20substrate%20specificity%20of%20FKBP35%20from%20Plasmodium%20Knowlesi%20using%20Protease-Free%20Assay%20abstract.pdf
https://eprints.ums.edu.my/id/eprint/27448/2/Reassessment%20of%20the%20Catalytic%20activity%20and%20substrate%20specificity%20of%20FKBP35%20from%20Plasmodium%20Knowlesi%20using%20Protease-Free%20Assay%20FULLTEXT.pdf
https://eprints.ums.edu.my/id/eprint/27448/
https://jurcon.ums.edu.my/ojums/index.php/bijb/article/view/2602
https://doi.org/10.51200/bijb.vi.2602
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score 13.211869

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