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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| Format: | Article |
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Universiti Malaysia Sabah
2020
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| author | Budiman, Cahyo Goh, Carlmond Kah Wun Lee, Ping Chin Razali, Rafida Thean, Chor Leow |
| author_facet | Budiman, Cahyo Goh, Carlmond Kah Wun Lee, Ping Chin Razali, Rafida Thean, Chor Leow |
| author_sort | Budiman, Cahyo |
| collection | UPM |
| 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. |
| first_indexed | 2024-03-06T10:43:36Z |
| format | Article |
| id | upm.eprints-87406 |
| institution | Universiti Putra Malaysia |
| last_indexed | 2024-03-06T10:43:36Z |
| publishDate | 2020 |
| publisher | Universiti Malaysia Sabah |
| record_format | dspace |
| spelling | 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 |
| 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 |
| title | 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_short | 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 |
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