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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: Budiman, Cahyo, Goh, Carlmond Kah Wun, Lee, Ping Chin, Razali, Rafida, Thean, Chor Leow
Format: Article
Published: Universiti Malaysia Sabah 2020
Online Access:http://psasir.upm.edu.my/id/eprint/87406/
https://jurcon.ums.edu.my/ojums/index.php/bijb/index
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spelling my.upm.eprints.874062024-01-12T08:06:38Z http://psasir.upm.edu.my/id/eprint/87406/ Reassessment of the catalytic activity and substrate specificity of fkbp35 from plasmodium knowlesi using protease-free assay Budiman, Cahyo Goh, Carlmond Kah Wun Lee, Ping Chin Razali, Rafida Thean, Chor Leow 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. Universiti Malaysia Sabah 2020-12 Article PeerReviewed Budiman, Cahyo and Goh, Carlmond Kah Wun and Lee, Ping Chin and Razali, Rafida 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/index 10.51200/bijb.v1i.2602
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
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 Budiman, Cahyo
Goh, Carlmond Kah Wun
Lee, Ping Chin
Razali, Rafida
Thean, Chor Leow
spellingShingle Budiman, Cahyo
Goh, Carlmond Kah Wun
Lee, Ping Chin
Razali, Rafida
Thean, Chor Leow
Reassessment of the catalytic activity and substrate specificity of fkbp35 from plasmodium knowlesi using protease-free assay
author_facet Budiman, Cahyo
Goh, Carlmond Kah Wun
Lee, Ping Chin
Razali, Rafida
Thean, Chor Leow
author_sort Budiman, Cahyo
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 Universiti Malaysia Sabah
publishDate 2020
url http://psasir.upm.edu.my/id/eprint/87406/
https://jurcon.ums.edu.my/ojums/index.php/bijb/index
_version_ 1789426937707364352
score 13.211869

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