• Cahyo Budiman Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu Sabah, Malaysia
  • Carlmond Goh Kah Wun Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu Sabah, Malaysia
  • Lee Ping Chin Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Kota Kinabalu Sabah, Malaysia
  • Rafida Razali Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu Sabah, Malaysia
  • Thean Chor Leow Enzyme and Microbial Technology Research Center, Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia



malaria, Plasmodium knowlesi, Peptidyl prolyl cis-trans isomerases, FKBP


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.

Author Biography

Rafida Razali, Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu Sabah, Malaysia


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How to Cite

Budiman, C., Goh Kah Wun, C. ., Lee Ping Chin, Razali, R. ., & 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), 125 - 143.